Hypophosphorylation Of Retinoblastoma Protein Research Articles

A novel role for α-viniferin in suppressing angiogenesis by blocking the VEGFR-2/p70S6K signaling pathway.

Angiogenesis plays important roles in pathological conditions such as cancer and inflammation as well as normal tissue development and homeostasis. Here, we investigated the effects and molecular mechanisms of α-viniferin, an oligostilbene isolated from Caragana sinica, on human umbilical vein endothelial cell responses in vitro and angiogenic sprouting in aortic rings ex vivo. α-viniferin treatment inhibited mitogen-induced HUVEC proliferation by retinoblastoma protein hypophosphorylation. In addition, α-viniferin suppressed mitogen-induced HUVEC adhesion, migration, invasion, and microvessel outgrowth. These anti-angiogenic activities of α-viniferin might be mediated through downregulation of cell cycle-related proteins, vascular endothelial growth factor receptor-2 (VEGFR-2), and matrix metalloproteinase-2. Furthermore, inactivation of VEGFR-2/p70 ribosomal S6 kinase signaling pathway was found to be involved in α-viniferin-mediated modulation of endothelial cell responses. Our results demonstrate the pharmacological functions and molecular mechanisms of α-viniferin in regulating angiogenesis, suggesting the therapeutic potential of α-viniferin to treat and prevent various angiogenesis-related diseases.

Sprouty1 Prevents Cellular Senescence Maintaining Proliferation and Differentiation Capacity of Human Adipose Stem/Progenitor Cells.

The role of Ras-Mitogen-activated protein kinase (MAPK) signaling in cellular aging is not precisely understood. Recently, we identified Sprouty1 (SPRY1) as a weight-loss target gene in human adipose stem/progenitor cells (ASCs) and showed that Sprouty1 is important for proper regulation of adipogenesis. In the present study, we show that loss-of-function of Sprouty1 by CRISPR/Cas9-mediated genome editing in human ASCs leads to hyper-activation of MAPK signaling and a senescence phenotype. Sprouty1 knockout ASCs undergo an irreversible cell cycle arrest, become enlarged and stain positive for senescence-associated β-galactosidase. Sprouty1 down-regulation leads to DNA double strand breaks, a considerably increased number of senescence-associated heterochromatin foci and induction of p53 and p21Cip1. In addition, we detect an increase of hypo-phosphorylated Retinoblastoma (Rb) protein in SPRY1 knockout ASCs. p16Ink4A is not induced. Moreover, we show that Sprouty1 knockout leads to induction of a senescence-associated secretory phenotype as indicated by the activation of the transcription factors NFκB and C/EBPβ and a significant increase in mRNA expression and secretion of interleukin-8 (IL-8) and CXCL1/GROα. Finally, we demonstrate that adipogenesis is abrogated in senescent SPRY1 knockout ASCs. In conclusion, this study reveals a novel mechanism showing the importance of Sprouty1 for the prevention of senescence and the maintenance of the proliferation and differentiation capacity of human ASCs.

Abstract A066: Rb and ZBTB7A cooperatively mediate the transcriptional repression function of androgen receptor

Abstract Prostate cancer (PCa) is one of the most common cancers in men. Primary PCa depends on androgen for growth and survival by binding to the C-terminal ligand-binding domain of androgen receptor (AR), a transcriptional factor that regulates multiple gene expression. The standard treatment of PCa is surgical or medical castration (known as androgen deprivation therapy, ADT) to reduce circulating androgens. However, patients invariably relapse into a more aggressive castration-resistant prostate cancer (CRPC) and AR remains important in this stage of disease. Although AR is well known for its transcriptional activator function, it can also suppress the expression of a subset of genes, including AR itself, multiple androgen synthetic genes, and a group of DNA replication genes. Mechanistically, we have shown that AR globally recruits hypophosphorylated retinoblastoma protein (Rb) to the promoters/enhancers of DNA replication gene loci and strengthens the activity of Rb-E2F suppressor complex. This novel activity of AR provides one molecular basis for treating CRPC patients with high level androgens. Importantly, we have also shown in vitro that this tumor-suppressor function of AR can be enhanced by CDK4/6 inhibitors, which block Rb phosphorylation. Although most CRPC are Rb-positive, recent sequencing studies indicate that ~10-15% of CRPC showed Rb loss in their chromosome. Therefore, we have generated tetracycline-inducible Rb-silencing CRPC cell lines to further examine the impact of Rb-loss on the transcriptional repression activity of AR. Consistently, we have found that Rb-silencing globally impaired the AR repression on DNA replication genes. Furthermore, we have also examined the AR-independent function of Rb and found that Rb-loss led to the activation of neuroendocrine genes. To further understand the mechanisms of how AR performs its transcriptional repression activity, we analyzed AR ChIP-seq data and androgen-regulated gene profile. This analysis resulted in the identification of ZBTB7A binding motif as the most enriched motif within AR repression sites. ZBTB7A, also known as LRF/POKEMON, is a zinc finger and BTB domain containing transcription repressor consisting of a protein-protein interacting BTB domain at N-terminus and DNA binding zinc fingers at C-terminus. Even though ZBTB7A has been identified as a proto-oncogene, a recent study using transgenic mouse models shows that it functions as a tumor suppressor in driving PCa progression with PTEN-null background. Therefore, we hypothesize that ZBTB7A might play a key role in regulating the Rb-dependent transcriptional repressor activity of AR. To globally determine this function of ZBTB7A, we carried out ZBTB7A ChIP-seq to identify high confident binding peaks. Motif analysis on these peaks indicated the marked enrichment of binding motif of ZBTB family members, suggesting that these binding sites are specific for ZBTB7A binding. Significantly, ~30% of these ZBTB7A binding sites overlapped with AR binding sites, particularly at promoter region. Using the unbiased Binding and Expression Target Analysis, we further found that these ZBTB7A and AR cobinding sites were significantly associated with the expression of AR-repressed genes. More importantly, the binding of ZBTB7A at these sites highly associated with Rb binding and silencing ZBTB7A impaired the AR repression activity on the DNA replication genes, suggesting that ZBTB7A may cooperate with Rb in regulating AR repressor activity. By coimmunoprecipitation assays, we found that ZBTB7A physically interacts with AR and Rb and these interactions are enhanced by androgen stimulation, suggesting that ZBTB7A is an additional component in the AR repressor complex. Furthermore, by additional ChIP-seq analyses we have also found that ZBTB7A binding at those AR repression sites was rapidly increased upon androgen stimulation, further indicating that AR may recruit ZBTB7A to the chromatin and ZBTB7A and Rb may cooperatively mediate the transcriptional activity of AR. This study provides novel insights in the molecular basis of transcriptional repression function of AR and may have significant therapeutic impact on enhancing current androgen therapy to treat CRPC patients by enhancing the expression or activity of ZBTB7A. Citation Format: Wanting Han, Dong Han, Sujun Chen, Shuai Gao, Housheng He, Changmeng Cai. Rb and ZBTB7A cooperatively mediate the transcriptional repression function of androgen receptor [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A066.

Trigonostemon reidioides modulates endothelial cell proliferation, migration and tube formation via downregulation of the Akt signaling pathway.

Trigonostemon reidi`oides (TR) is used as a Thai traditional medicine for the treatment of drug addiction, asthma, food poisoning, constipation and snake bites. The present study investigated the effects and molecular mechanisms of the ethanolic extract of TR (ETR) on mitogen-induced human umbilical vein endothelial cells (HUVECs) responses, proliferation, adhesion, migration and tube formation. ETR treatment inhibited mitogen-induced HUVEC proliferation by downregulation of cell cycle-associated proteins, including cyclins and cyclin-dependent kinases, which induced retinoblastoma protein hypophosphorylation. The present study also demonstrated that ETR treatment suppressed mitogen-induced HUVEC adhesion, migration, invasion and tube formation, and that these anti-angiogenic activities were mediated by inactivation of mitogen-induced Akt and matrix metalloproteinase (MMP)-2, but not of extracellular signal-regulated kinase, p70 ribosomal S6 kinase or MMP-9. Collectively, the results of the present study suggested pharmacological functions and molecular mechanisms of ETR in regulating endothelial cell fates, and supported further evaluation and development of ETR as a potential therapeutic agent for the treatment and prevention of angiogenesis-associated diseases, including cancer.

Research Article: Simian virus 40 large T antigen-mediated transactivation of the cyclin A promoter is independent of p53 and TBP binding functions

Abstract Simian virus 40 is a small DNA tumor virus capable of transforming cells in culture and causing tumors in animal models. The viral protein large T antigen specifically binds the tumor suppressors p53 and hypophosphorylated retinoblastoma protein (pRb) to prevent G1 arrest. T antigen also transactivates the cyclin-A promoter. This activity is independent of pRb binding, yet dependent upon the J-domain Hsp70-binding region. T antigen also binds transcription factors, including TATA box binding protein (TBP). Thus, we assessed whether cyclin A promoter activation relied on this activity and/or the ability to complex with p53. Using a luciferase reporter assay, we show that T antigen mutant proteins defective in TBP and p53 binding are able to transactivate the cyclin A promoter to wild type levels. These data indicate that T antigen mediated transactivation of the cyclin A promoter uses a p53 and TBP-binding-independent mechanism, yet relies on hsp70 binding to the N-terminal, J-domain. This suggest...

Androgen Receptor Tumor Suppressor Function Is Mediated by Recruitment of Retinoblastoma Protein

Although well characterized as a transcriptional activator that drives prostate cancer (PCa) growth, androgen receptor (AR) can function as a transcriptional repressor, and high-level androgens can suppress PCa proliferation. The molecular basis for this repression activity remains to be determined. Genes required for DNA replication are highly enriched among androgen-repressed genes, and AR is recruited to the majority of these genes, where it rapidly represses their transcription. This activity is enhanced in PCa cells expressing high AR levels and is mediated by recruitment of hypophosphorylated retinoblastoma protein (Rb). Significantly, AR also indirectly increases the expression of DNA replication genes through stimulatory effects on other metabolic genes with subsequent CDK activation and Rb hyperphosphorylation. In castration-resistant PCa cells, which are dependent on high-level AR expression, this anti-proliferative repression function might be exploited through treatment withandrogen in combination with agents that suppress AR-driven metabolic functions or cell cycle progression.

Long-term HIV-1 infection induces an antiviral state in primary macrophages

HIV-1 infection is thought to impair type I interferon (IFN-I) production in macrophages, a cell type that is also relatively resistant to HIV-1 cytotoxic effects. Here, we show that monocyte differentiation into macrophages by M-CSF led to cell proliferation and susceptibility to HIV-1 infection that induced cell cycle arrest and increased cell death. Established HIV-1 infection of monocyte-derived macrophages induced the upregulation of the pattern recognition receptors MDA5 and Rig-I that serve as virus sensors; production of interferon-β, and transcription of interferon-stimulated genes including CXCL10. Infected macrophages showed increased expression of p21 and subsequent inactivation of cyclin-CDK2 activity leading to a hypo-phosphorylated active retinoblastoma protein (pRb) and deactivation of E2F1-dependent transcription and CDK1 downregulation. Additionally, HIV-1 infection limited deoxynucleotide pool by downregulation of the ribonucleotide reductase subunit R2 (RNR2) and reactivation of the HIV-1 restriction factor SAMHD1 together with increased cell death. In conclusion, HIV-1 induced an innate antiviral mechanism associated to IFN-I production, interferon stimulated gene activation, and p21-mediated G2/M arrest leading to elevated levels of cell death in monocyte derived macrophages. Upregulation of MDA5 and Rig-I may serve as targets for the development of antiviral strategies leading to the elimination of HIV-1 infected cells.

Abstract 2477: An orally bioavailable and selective histone deacetylase (HDAC) 1 & 2 inhibitor enhances retinoic acid mediated differentiation of neuroblastoma

Abstract INTRODUCTION: Neuroblastoma (NB) is an extra-cranial solid cancer and is among the most common cancers in infants less than 1 year of age, with 650 new cases each year in the United States. Half of the children with NB have high risk disease and 20-50% of those will fail to respond adequately to current therapies, illustrating an urgent unmet medical need. Current treatment for high-risk disease is aggressive, including chemotherapy, surgery, radiation with stem cell transplant, anti-GD2/cytokine immunotherapy and retinoic acid (RA) treatment. RA is a pro-differentiation agent that slows growth and promotes cell death. A gene expression pattern associated with RA-induced NB differentiation was identified, and chemical inhibition of HDAC1/2 was shown to induce a similar expression pattern. METHODS: In this work, we examine the activity of an orally bioavailable HDAC1/2 inhibitor (HDAC1/2i) on NB cell differentiation, proliferation and apoptosis. RA combined with HDAC1/2i enhances gene expression patterns associated with differentiation, slows cellular proliferation and more rapidly induces dendrite formation than RA can achieve alone. The mechanisms leading to the differentiated phenotype were examined by RT-PCR, gene expression microarray and retinoic acid receptor (RAR) chromatin immunoprecipitation followed by high-througput sequening (ChIP-Seq). RESULTS: HDAC1/2i and RA together caused increased localization of the RAR to its own RARα and RARβ promoter regions, and increased in RAR mRNA and protein relative to the RA treatment condition alone. Additionally, expression of Cyp26, an enzyme responsible for clearing intercellular RA, was reduced in the combination setting. Gene set enrichment analysis of the microarray data comparing the combination setting against RA as a single agent suggested that the addition of HDAC1/2i enhanced apoptotic pathways and decreased E2F driven cell cycle signaling. We confirmed enhanced apoptosis in the combination setting by measuring caspase 3 and PARP cleavage. Consistent with this finding, we observed reduced proliferation, increased sub-G1 cell frequency in cell cycle assays and ablation of emergent RA-resistant NB colonies after combination treatment. Further, combination treatment reduced the E2F-activators CDK4 and CDK6 at the protein level while the CDK inhibitor, p21, was dramatically increased. Hypo-phosphorylation of retinoblastoma protein, directly linked to E2F complex inactivation, was also observed and consistent with reduced proliferation and the decreased frequency of S-phase cells observed in EDU incorporation assays. Xenograft models of NB with RA and HDAC1/2i are in progress, as are HDAC1, HDAC2, acetylated histone H3K9 ChIP-seq experiments, and will be discussed. Taken together, these findings support a role for selective HDAC1/2i in combination with RA for the treatment of patients with high risk NB. Citation Format: David L. Tamang, Pengyu Huang, Olga Golonzhka, Jeffrey R. Shearstone, Steven N. Quayle, Simon S. Jones, Min Yang. An orally bioavailable and selective histone deacetylase (HDAC) 1 & 2 inhibitor enhances retinoic acid mediated differentiation of neuroblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2477.

Cyclin D type does not influence cell cycle response to DNA damage caused by ionizing radiation in multiple myeloma tumours.

Multiple myeloma (MM) is characterized by over-expression of cyclin D1 (CCND1) or D2 (CCND2), which control G1 phase cell-cycle progression. Proteolytic degradation of CCND1 (but not CCND2), resulting in G1 arrest, is reported in non-MM cells post-DNA damage, affecting DNA repair and survival. We examined the effect of ionizing radiation (IR) on D-cyclin levels and cell-cycle kinetics of MM cells, exploring differences based on D-cyclin expression. We showed that CCND1 is downregulated, whereas CCND2 is not, following IR. This did not lead to hypo-phosphorylation of retinoblastoma protein or G1 arrest. Both CCND1- and CCND2-expressing MM cells arrested in S/G2/M, and did not differ in other cell-cycle proteins or sensitivity to IR. When treated with a CDK4/6 inhibitor, both CCND1 and CCND2 MM cells arrested in G1 and therefore are subject to physiological regulation at this checkpoint. Immunoprecipitation showed that, despite CCND1 degradation following IR, sufficient protein remains bound to CDK4/6 to prevent G1 arrest. Aberrant expression of CCND1 driven from the IGH promoter in t(11;14) MM cells maintains progression through G1 to arrest in S/G2/M. Differential expression of D-cyclin does not appear to affect cell-cycle response to IR, and is unlikely to underlie differential sensitivity to DNA damage.

Abstract A136: A novel, orally bioavailable and selective histone deacetylase (HDAC) 1 & 2 inhibitor enhances retinoic acid mediated differentiation of neuroblastoma

Abstract Neuroblastoma (NB) is an extra-cranial solid cancer arising from the neural crest and is among the most common cancers in infants less than 1 year of age (Park, JR et al., 2008). Approximately one child per 100,000 is diagnosed with NB, resulting in 650 new cases each year in the United States. Half of the children with NB have high risk disease and 20-50% of those will fail to respond adequately to current therapies, illustrating a clear unmet medical need. Current treatment for high-risk disease is aggressive, including chemotherapy, surgery, radiation with stem cell transplant, anti-GD2/cytokine immunotherapy and retinoic acid (RA) (Yang, RK et. al., 2010; Cheung, NK et. al., 2012). RA is a pro-differentiation agent that slows growth and promotes cell death. A gene expression pattern associated with RA-induced NB differentiation was identified (Hahn, CK et. al., 2008; Frumm, SM et. al., 2013), and inhibition of HDAC1/2 was shown to induce a similar expression pattern. In this work, we examine the activity of an orally bioavailable HDAC1/2 inhibitor (HDAC1/2i) on NB cell differentiation, proliferation and apoptosis. RA combined with HDAC1/2i enhances gene expression patterns associated with differentiation, slows cellular proliferation and more rapidly induces dendrite formation than RA can achieve alone. The mechanisms leading to the differentiated phenotype were examined by microarray and retinoic acid receptor (RAR) ChIP-seq. HDAC1/2i and RA together caused increased localization of the RAR to its own RARa and RARß promoter regions, and an increase in mRNA and protein relative to the RA treatment condition alone. Additionally, expression of Cyp26a1/b1, enzymes responsible for clearing intercellular RA, were reduced in the combination setting. Gene set enrichment analysis of the microarray data comparing the combination setting against RA as a single agent suggested that the addition of HDAC1/2i was enhancing apoptotic pathways and decreasing E2F driven cell cycle signaling. In further experiments, we confirmed enhanced apoptosis in the combination setting by measuring caspase 3 and PARP cleavage, which is consistent with reduced proliferation, increased sub-G1 cell frequency in cell cycle assays and ablation of emergent RA-resistant NB colonies. Further, the E2F-activators, CDK4 and CDK6, were reduced at the protein level in the combination setting while the CDK inhibitor, p21, was dramatically increased. Hypo-phosphorylation of retinoblastoma protein, directly linked to E2F complex inactivation, was also observed and consistent with reduced proliferation and the decreased frequency of S-phase cells observed in EDU incorporation assays. Xenograft models of NB with RA and HDAC1/2i are in progress and will be discussed. Taken together, these findings support a role for selective HDAC1/2i in combination with RA for the treatment of patients with high risk NB. Citation Format: David L. Tamang, Pengyu Haung, Olga Golonzhka, Jeff Shearstone, Steven N. Quayle, Simon S. Jones, Min Yang. A novel, orally bioavailable and selective histone deacetylase (HDAC) 1 & 2 inhibitor enhances retinoic acid mediated differentiation of neuroblastoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A136.

The direct and indirect effects of corticosterone and primary adipose tissue on MCF7 breast cancer cell cycle progression.

Breast cancer is the second leading cause of cancer-related mortality in women. Glucocorticoids (GCs) have the potential to directly affect breast cancer or indirectly via changes to the tumor growth microenvironment a breast cancer is exposed to. The role of GCs in breast cancer progression by direct and indirect means are not fully understood. To study the direct and indirect effects of GCs on breast cancer cell cycle regulation. MCF7 breast cancer cells were incubated with increasing concentrations of corticosterone (CORT) to investigate the direct effects. In addition, MCF7 cells were cultured in conditioned media (CM) from primary adipose tissue excised from CORT-supplemented lean and obese male rats. CORT alone resulted in dose-dependent increases in p27 and hypophosphorylated retinoblastoma protein (Rb) which was accompanied by a reduction in the number of cells in S-phase. CM prepared from adipose tissue overrode these direct CORT effects, suggesting that the tumor growth microenvironment created in the CM dominates MCF7 cell cycle regulation. The direct inhibitory effects of CORT on cancer cell cycle progression are largely limited by the hormone's effects on adipose tissue biology.

Targeting Nedd8 Activating Enzyme Induces DNA Damage and Cell Cycle Arrest and Sensitizes Chronic Lymphocytic Leukemia (CLL) B-Cells to Alkylating Agents

Microenvironment-mediated upregulation of the B-cell receptor (BCR) and nuclear factor-kappaB (NFκB) signaling in the CLL cells resident in the lymph node and bone marrow promotes apoptosis evasion and clonal expansion. We recently reported that stromal-mediated NFκB pathway activity and CLL cell survival may be successfully abrogated in vitrousing MLN4924. MLN4924 is an investigational agent that inhibits the NEDD8-activating enzyme (NAE) and thereby prevents neddylation of Cullin-RING ubiquitin ligases (CRL), resulting in stabilization of their protein substrates, including inhibitor of NFκB. However, targeting NAE also slows degradation of other proteins and thus full biological consequences are tissue-dependent. In adherent solid tumor cell lines, MLN4924 induced expression of Cdt1, a DNA replication licensing factor, followed by DNA damage and cell cycle arrest, but the importance of this mechanism in primary neoplastic B-cells has not been determined. Here we show that, under the growth conditions, targeting NAE induces DNA damage and cell cycle arrest in primary CLL B-cells.B-cells obtained from patients with CLL were co-cultured in vitro with CD40L-expressing stroma to mimic the pro-survival conditions present in lymphoid tissue. CLL cells were incubated in the presence or absence of 25 ng/mL IL-21. MLN4924 was provided by Millennium Pharmaceuticals, Inc.CD40L-expressing stromal co-cultures rapidly induced anti-apoptotic BCL2 family proteins MCL1 and BCLX in peripheral blood CLL cells, leading to protection from spontaneous apoptosis and chemoresistance. Prolonged (72 h) co-cultures induced cell cycle progression in ~5% of the CLL cells. IL-21 further enhanced proliferation and sensitized CLL cells to MLN4924. Thus, treatment with 1 μM MLN4924 induced apoptosis in 44.6±5.4% of the CD40L-stimulated, and in 63.7±2.6% of the CD40L/IL-21-stimulated CLL cells at 24 h (p=0.00004). CD40-stimulated CLL cells treated with MLN4924 showed Cdt1 accumulation, DNA re-replication, activation of DNA damage response pathways as evidenced by accumulation of pRPA, pChk1, pChk2 and gH2AX which preceded PARP cleavage, and G2 arrest. DNA damage and G2 arrest were more prominent in IL-21-treated cells. Targeting NAE also resulted in early accumulation of the endogenous cyclin-dependent kinase inhibitors p21 and p27, both CRL targets. By contrast, retinoblastoma protein hypophosphorylation occurred late and did not precede apoptosis. siRNA-mediated knockdown of Cdt1, but not p21 or p27 prevented MLN4924-mediated DNA damage response and CLL cell apoptosis. Finally, MLN4924 did not lead to accumulation of Cdt1, p21 or p27 in CLL cells cultured off stroma.Since alkylating agents target primarily cycling cells, we further determined whether they would cooperate with MLN4924 to induce DNA damage in CLL. While either chlorambucil or bendamustine exhibited little toxicity against the CD40L-stimulated CLL cells, MLN4924 sensitized CLL cells to those agents. MLN4924 further augmented bendamustine toxicity in the presence of IL-21. MLN4924-mediated DNA damage response was augmented in the presence of bendamustine. Interestingly, this combination proved ineffective in the CLL samples with del(17p), suggesting that bendamustine effect depends on the intact p53 pathway.Here we characterize DNA damage response pathway in CLL, where it remains understudied. Targeting NAE in cycling CLL cells induces Cdt1 accumulation, DNA damage and cell cycle arrest. This data provide additional insights into the biological consequences of targeting NAE in CLL B-cells as well as preclinical rationale to study the potential clinical activity of MLN4924 in CLL, alone or in combination with bendamustine or chlorambucil. DisclosuresBerger:Takeda: Employment.

Inhibition of Cancer Cell Proliferation by PPARγ Is Mediated by a Metabolic Switch that Increases Reactive Oxygen Species Levels

The nuclear receptor peroxisome-proliferation-activated receptor gamma (PPARγ), a transcriptional master regulator of glucose and lipid metabolism, inhibits the growth of several common cancers, including lung cancer. In this study, we show that the mechanism by which activation of PPARγ inhibits proliferation of lung cancer cells is based on metabolic changes. We found that treatment with the PPARγ agonist pioglitazone triggers a metabolic switch that inhibits pyruvate oxidation and reduces glutathione levels. These PPARγ-induced metabolic changes result in a marked increase of reactive oxygen species (ROS) levels that lead to rapid hypophosphorylation of retinoblastoma protein (RB) and cell-cycle arrest. The antiproliferative effect of PPARγ activation can be prevented by suppressing pyruvate dehydrogenase kinase 4 (PDK4) or β-oxidation of fatty acids in vitro and in vivo. Our proposed mechanism also suggests that metabolic changes can rapidly and directly inhibit cell-cycle progression of cancer cells by altering ROS levels.

Cisplatin Plus Sodium Arsenite and Hyperthermia Induces Pseudo-G1 Associated Apoptotic Cell Death in Ovarian Cancer Cells

Cisplatin is effective against solid tumors including ovarian cancer. However, inherent or acquired cisplatin resistance limits clinical success. We recently demonstrated that a combination of sodium arsenite (NaAsO2) and hyperthermia sensitizes p53-expressing ovarian cancer cells to cisplatin by modulating DNA repair pathway and enhancing platinum accumulation. However, it is not understood how this combination therapy modulates cell cycle following platinum-DNA damage. The goal of the present study was to determine if NaAsO2 and hyperthermia alter cisplatin-induced G2 arrest and cause mitotic arrest and mitotic catastrophe. Human epithelial ovarian cancer cells (A2780 and A2780/CP70) were treated with cisplatin ± 20 μM NaAsO2 at 37 or 39°C for 1 h. Cisplatin ± NaAsO2 at 37 or 39°C caused cells to accumulate in G2/M compartment at 36 h after treatment. Western blot analysis of cyclin A and cyclin B suggested that combined NaAsO2, hyperthermia, and cisplatin induced mitotic arrest. However, we observed < 3% mitotic index and phosphorylation of histone H3 on serine 10 was undetectable. These results did not confirm mitotic arrest. BUBR1 (BUB1B) also was not phosphorylated, suggesting disrupted mitotic checkpoint. Postmitotic cells accumulated in pseudo-G1 as demonstrated by cyclin E stabilization, CDKN1A induction, and hypophosphorylation of retinoblastoma protein. These cells also were positive for Annexin V binding indicating they were apoptotic. In summary, cisplatin plus NaAsO2 and hyperthermia induced pseudo-G1 associated apoptosis in ovarian cancer cells.

Silencing of protein kinase D2 induces glioma cell senescence via p53-dependent and -independent pathways

BackgroundGlioblastoma multiforme (GBM) is a highly aggressive tumor of the central nervous system with a dismal prognosis for affected patients. Aberrant protein kinase C (PKC) signaling has been implicated in gliomagenesis, and a member of the PKC-activated protein kinase D (PRKD) family, PRKD2, was identified as mediator of GBM growth in vitro and in vivo.MethodsThe outcome of PRKD2 silencing and pharmacological inhibition on glioma cell proliferation was established with different glioma cell lines. Western blotting, senescence assays, co-immunoprecipitation, fluorescence activated cell sorting, quantitative PCR, and immunofluorescence microscopy were utilized to analyze downstream signaling.ResultsRNA-interference (21-mer siRNA) and pharmacological inhibition (CRT0066101) of PRKD2 profoundly inhibited proliferation of p53wt (U87MG, A172, and primary GBM2), and p53mut (GM133, T98G, U251, and primary Gli25) glioma cells. In a xenograft experiment, PRKD2 silencing significantly delayed tumor growth of U87MG cells. PRKD2 silencing in p53wt and p53mut cells was associated with typical hallmarks of senescence and cell cycle arrest in G1. Attenuated AKT/PKB phosphorylation in response to PRKD2 silencing was a common observation made in p53wt and p53mut GBM cells. PRKD2 knockdown in p53wt cells induced upregulation of p53, p21, and p27 expression, decreased phosphorylation of CDK2 and/or CDK4, hypophosphorylation of retinoblastoma protein (pRb), and reduced transcription of E2F1. In p53mut GM133 and primary Gli25 cells, PRKD2 silencing increased p27 and p15 and reduced E2F1 transcription but did not affect pRb phosphorylation.ConclusionsPRKD2 silencing induces glioma cell senescence via p53-dependent and -independent pathways.

Aging increases CCN1 expression leading to muscle senescence

Using microarray analysis, we found that aging sarcopenia is associated with a sharp increase in the mRNA of the matricellular protein CCN1 (Cyr61/CTGF/Nov). CCN1 mRNA was upregulated 113-fold in muscle of aged vs. young rats. CCN1 protein was increased in aging muscle in both rats (2.8-fold) and mice (3.8-fold). When muscle progenitor cells (MPCs) were treated with recombinant CCN1, cell proliferation was decreased but there was no change in the myogenic marker myoD. However, the CCN1-treated MPCs did express a senescence marker (SA-βgal). Interestingly, we found CCN1 increased p53, p16(Ink4A), and pRP (hypophosphorylated retinoblastoma protein) protein levels, all of which can arrest cell growth in MPCs. When MPCs were treated with aged rodent serum CCN1 mRNA increased by sevenfold and protein increased by threefold suggesting the presence of a circulating regulator. Therefore, we looked for a circulating regulator. Wnt-3a, a stimulator of CCN1 expression, was increased in serum from elderly humans (2.6-fold) and aged rodents (2.0-fold) compared with young controls. We transduced C2C12 myoblasts with wnt-3a and found that CCN1 protein was increased in a time- and dose-dependent manner. We conclude that in aging muscle, the circulating factor wnt-3a acts to increase CCN1 expression, prompting muscle senescence by activating cell arrest proteins.

AMXT-1501, a novel polyamine transport inhibitor, synergizes with DFMO in inhibiting neuroblastoma cell proliferation by targeting both ornithine decarboxylase and polyamine transport

Neuroblastoma (NB) is associated with MYCN oncogene amplification occurring in approximately 30% of NBs and is associated with poor prognosis. MYCN is linked to a number of genes including ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis. ODC expression is elevated in many forms of cancer including NB. Alpha-difluoromethylornithine (DFMO), an ODC inhibitor, is currently being used in a Phase I clinical trial for treatment of NB. However, cancer cells treated with DFMO may overcome their polyamine depletion by the uptake of polyamines from extracellular sources. A novel polyamine transport inhibitor, AMXT-1501, has not yet been tested in NB. We propose that inhibiting ODC with DFMO, coupled with polyamine transport inhibition by AMXT-1501 will result in enhanced NB growth inhibition. Single and combination drug treatments were conducted on three NB cell lines. DFMO IC50 values ranged from 20.76 to 33.3 mM, and AMXT-1501 IC50 values ranged from 14.13 to 17.72 µM in NB. The combination treatment resulted in hypophosphorylation of retinoblastoma protein (Rb), suggesting growth inhibition via G1 cell cycle arrest. Increased expression of cleaved PARP and cleaved caspase 3 in combination-treated cells starting at 48 hr suggested apoptosis. The combination treatment depleted intracellular polyamine pools and decreased intracellular ATP, further verifying growth inhibition. Given the current lack of effective therapies for patients with relapsed/refractory NB and the preclinical effectiveness of DFMO with AMXT-1501, this combination treatment provides promising preclinical results. DFMO and AMXT-1501 may be a potential new therapy for children with NB.

Abstract 1743: SOX10 is required for cell cycle regulation and melanomagenesis.

Abstract The transcription factor SOX10 is essential for proper survival and differentiation of neural crest cell lineages, and plays an important role in the establishment and maintenance of melanocytes. SOX10 is also highly expressed in melanoma tumors, however its role in the progression of the disease is not yet understood. Here, we report that melanoma tumor cell lines require wild-type SOX10 expression for proliferation, and detail the rescue of melanoma in the metabotropic glutamate receptor 1 (Grm1Tg) transgenic mouse by Sox10 haploinsufficiency. Stable SOX10 knockdown in human melanoma cells results in arrested growth, altered cellular morphology, and senescence. Cells with a stable loss of SOX10 are arrested in the G1 phase of the cell cycle with elevated cyclin-dependent kinase inhibitor 1A and 1B (p21 and p27) expression. SOX10 loss also results in hypophosphorylation of retinoblastoma protein (RB) and a reduction in the expression of its binding partner E2F1. Previously, the RB-E2F1 pathway has been shown to play a central role in cell cycle regulation, and here we demonstrate that SOX10 expression is required for the maintenance of the RB-E2F1 pathway to promote melanoma cell growth. One of the main events driving cellular transformation is the dysregulation of the cell cycle, therefore the role that SOX10 has in maintaining this process provides great promise for targeted interventions. Citation Format: Julia C. Cronin, Dawn E. Watkins-Chow, Art Incao, Joanne H. Hasskamp, Nicola Schönewolf, Nicholas K. Hayward, Boris C. Bastian, Reinhard Dummer, Stacie K. Loftus, William J. Pavan. SOX10 is required for cell cycle regulation and melanomagenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1743. doi:10.1158/1538-7445.AM2013-1743

Mechanisms of Resveratrol-Induced Inhibition of Clonal Expansion and Terminal Adipogenic Differentiation in 3T3-L1 Preadipocytes

We show that resveratrol prevents clonal expansion and terminal adipogenesis in 3T3-L1 preadipocytes. An early resveratrol effect was the inhibition of AKT and mitogen-activated protein kinase signaling, accompanied by down regulation of cyclin D1 expression, abrogation of retinoblastoma protein hyperphosphorylation, and subsequent inhibition of cell cycle reentry and clonal expansion, as indicated by cyclin A2 repression. Resveratrol inhibited terminal adipogenesis at the level of peroxisome proliferator-activated receptor-γ2 expression and activity. This was independent from the preceding inhibition of clonal expansion. Peroxisome proliferator-activated receptor-γ2 overexpression and activation partially restored fatty acid-binding protein 4 induction in resveratrol-treated 3T3-L1. Resveratrol activated AMP-activated protein kinase (AMPK) but did not induce PPAR-γ co-activator 1α (PGC1α) and mitochondrial biogenesis in 3T3-L1. Treatment with the Sirt1 inhibitor splitomicin augmented downregulation of peroxisome proliferator-activated receptor-γ2 and fatty acid-binding protein 4 expressions in resveratrol-treated 3T3-L1 and did not prevent the inhibition of terminal adipogenesis. Moreover, splitomicin could not obviate resveratrol-induced cyclin D1 repression, retinoblastoma protein hypophosphorylation, and inhibition of clonal expansion. Our data suggest that resveratrol inhibits clonal expansion and terminal adipogenesis in 3T3-L1 by several mechanisms.

A novel CyclinE/CyclinA-CDK Inhibitor targets p27Kip1 degradation, cell cycle progression and cell survival: Implications in cancer therapy

p27Kip1 (p27) binds and inhibits the cyclin E- or cyclin A-associated cyclin-dependent kinases (CDKs)2 and other CDKs, and negatively regulates G1–G2 cell cycle progression. To develop specific CDK inhibitors, we have modeled the interaction between p27 and cyclin A-CDK2, and designed a novel compound that mimics p27 binding to cyclin A-CDK2. The chemically synthesized inhibitor exhibited high potency and selective inhibition towards cyclin E/cyclin A-CDK2 kinase in vitro but not other kinases. To facilitate permeability of the inhibitor, a cell penetrating peptide (CPP) was conjugated to the inhibitor to examine its effect in several cancer cell lines. The CPP-conjugated inhibitor significantly inhibited the proliferation of cancer cells. The treatment of the inhibitor resulted in the increased accumulation of p27 and p21Cip1/Waf1 (p21) and hypo-phosphorylation of retinoblastoma protein (Rb). The degradation of p27, mediated through the phosphorylation of threonine-187 in p27, was also inhibited. Consequently, exposure of cells to the inhibitor caused cell cycle arrest and apoptosis. We conclude that specific cyclinE/cyclin A-CDK2 inhibitors can be developed based on the interaction between p27 and cyclin/CDK to block cell cycle progression to prevent tumor growth and survival.