P21 Cip1 Expression Research Articles

Abstract 559: The molecular kinetics of p21 and Cdk4 dictates irreversible G0/G1 phase cell cycle arrest in CRPC cells.

Abstract Deregulation of cell cycle progression is the hallmark of cancer; to this effect, several small molecules (AG-024322, AT7519, E7070, P1446A-05 and Flavopiridol) against cell cycle kinases are being developed to regulate cell cycle machinery that resulted in inhibition of tumor growth. The present study examined the effect of 3, 9-dihydroxy-2-prenylcoumestan-a furanocoumarin (Pso) on castration resistant prostate cancer cells (CRPC) cells. Treatment of CRPC (PC-3 and C4-2B) cells with Pso significantly caused irreversible G0-G1 cell cycle arrest that resulted in inhibition of cell proliferation. Protein analysis suggested that the expression of cdk inhibitors (CDKI) p21cip1 and p27kip1 were upregulated in both CRPC cell lines by Pso treatment. Studies using real-time reverse transcription-polymerase chain reaction analysis suggest that Pso transcriptionally regulates p21cip1 and p27kip1 in CRPC cells. Our immunofluorescence analysis pointed out that both p21cip1 and p27kip1 accumulated in the nucleus upon the treatment as compared to untreated CRPC cells. In addition, the cell cycle arrest was associated with decreased levels of both cyclins and cyclin-dependent kinases. However, cdk-4 appears to be more significantly downregulated when compared with either cyclins (D1, D2, D3 and Cyclin E) or other Cdk's (cdk2/6). Also, we noted an increased hypophosphorylated level of retinoblastoma (Rb) with a decreased in Rb phosphorylation resulted in inhibition of E2F1 function. Further, transient over expression of Cdk4 overcomes the Pso-induced G0-G1 arrest in CRPC cells. Also, knockdown of p21cip1 and p27kip1 expression with small interfering RNA significantly abrogated the Pso-induced G0-G1 cell cycle arrest suggested that induction of p21cip1 and p27kip1 is required for G0-G1 cell cycle arrest by Pso. Altogether our results strongly suggest that Pso could be a potential candidate for prevention and therapy against CRPC. Our ongoing studies using xenograft models of CRPC in nude mice may reveal the in vivo efficacy of Pso. Citation Format: Thippeswamy Gulappa, Rama S. Reddy, Suman Suman, Chendil Damodaran. The molecular kinetics of p21 and Cdk4 dictates irreversible G0/G1 phase cell cycle arrest in CRPC cells. [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 559. doi:10.1158/1538-7445.AM2013-559

Reactive oxygen species-mediated activation of the Akt/ASK1/p38 signaling cascade and p21Cip1 downregulation are required for shikonin-induced apoptosis

Shikonin derivatives exert powerful cytotoxic effects, induce apoptosis and escape multidrug resistance in cancer. However, the diverse mechanisms underlying their anticancer activities are not completely understood. Here, we demonstrated that shikonin-induced apoptosis is caused by reactive oxygen species (ROS)-mediated activation of Akt/ASK1/p38 mitogen-activated protein kinase (MAPK) and downregulation of p21(Cip1). In the presence of shikonin, inactivation of Akt caused apoptosis signal-regulating kinase 1 (ASK1) dephosphorylation at Ser83, which is associated with ASK1 activation. Shikonin-induced apoptosis was enhanced by inhibition of Akt, whereas overexpression of constitutively active Akt prevented apoptosis through modulating ASK1 phosphorylation. Silencing ASK1 and MKK3/6 by siRNA reduced the activation of MAPK kinases (MKK) 3/6 and p38 MAPK, and apoptosis, respectively. Antioxidant N-acetyl cysteine attenuated ASK1 dephosphorylation and p38 MAPK activation, indicating that shikonin-induced ROS is involved in the activation of Akt/ASK1/p38 pathway. Expression of p21(Cip1) was significantly induced in early response, but gradually decreased by prolonged exposure to shikonin. Overexpression of p21(Cip1) have kept cells longer in G1 phase and attenuated shikonin-induced apoptosis. Depletion of p21(Cip1) facilitated shikonin-induced apoptosis, implying that p21(Cip1) delayed shikonin-induced apoptosis via G1 arrest. Immunohistochemistry and in vitro binding assays showed transiently altered localization of p21(Cip1) to the cytoplasm by shikonin, which was blocked by Akt inhibition. The cytoplasmic p21(Cip1) actually binds to and inhibits the activity of ASK1, regulating the cell cycle progression at G1. These findings suggest that shikonin-induced ROS activated ASK1 by decreasing Ser83 phosphorylation and by dissociation of the negative regulator p21(Cip1), leading to p38 MAPK activation, and finally, promoting apoptosis.

Artemisia princeps var. orientalis induces cell cycle arrest accompanied by upregulation of p21CIP1 and caspase‐dependent apoptosis in human breast carcinoma cells

The aim of the present study was to investigate the anticancer activity and mechanism of action of methanol extract from Artemisia princeps var. orientalis (APME). First, the antiproliferative activity of APME against T47D cells was at various concentrations (5–200 μg/mL) and treatment times (24–72 h) determined by MTT assay. APME significantly inhibited cell proliferation in a dose‐dependent manner, regardless of treatment times. Under the same condition, cytotoxicity (LDH release) of APME was only appeared at 72 h. In addition, treatment with the IC50 concentration of APME for 24 h caused cell cycle arrest and apoptosis. APME arrested cells in both G1 and G2/M phases of the cell cycle, a response that was related to the induction of CDK1, cyclin B, p21Cip1 expression, and repression of CDK2 and cyclin A. The induction of p21Cip1 by APME caused activation of p53 and modulation of its downstream targets Bcl‐2 and Bax. Subsequently, caspase‐8 and ‐9 were cleaved in cells treated with APME. Activation of these caspases in turn led to increased activation of caspase‐3. The results of this study suggest that APME produce anticancer effects in T47D cells through cell cycle arrest associated with the induction of p21Cip1 and apoptosis mediated by p53 and caspase‐depedent pathways.This research was supported by Basic Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2012–0006811)

P38α and p38δ MAPK are key regulators of p21Cip1 gene expression in human keratinocytes

PKCδ increases keratinocyte differentiation via a mechanism that involves activation of p38δ MAPK. We recently showed that PKCδ also suppresses cell proliferation by increasing expression of the p21Cip1 cyclin‐dependent kinase inhibitor. However, the downstream effectors that mediate this PKCδ‐dependent regulation are not known. Our present studies suggest that PKCδ activates p38δ which regulates p21Cip1 promoter activity, as exogenously expressed p38δ increases p21Cip1 mRNA and protein level in human keratinocytes. Treatment with SB202190, a p38α/β inhibitor, or dominant negative p38α, leads to a decrease in PKCδ‐dependent p21Cip1 promoter activity, implicating p38α as a second mediator of this regulation. p21Cip1 promoter truncation experiments indicate that p38δ increases the activity via a putative p53 response element. This suggests that p38α and δ may act in conjunction with or through p53 to regulate p21Cip1 gene expression. In addition, we show that p38α and p38δ overexpression increases p53 promoter activity, suggesting that p38α and δ regulate p53 gene expression. We propose that PKCδ activates p38α and p38δ which in turn activates p53 to increase p21Cip1 expression leading to cessation of keratinocyte proliferation.

PDT Targeting Pulmonary Hypertension: Implications Prevention and Treatment by Photodynamic Therapy

Purpose Pulmonary arterial hypertension (PH) is an unremitting disease defined by progressive increase in pulmonary vascular resistance and vascular remodeling. Currently, little is known about treatments that affect vascular endothelial cell related pathogenic feature of PH. We propose to use photodynamic therapy (PDT) to treat PH, which use vascular specific light sensitive drug (a photosensitizer) and light in the presence of oxygen to irreversibly damage cells. We hypothesize that PDT can reverse vascular remodeling in PH through anti-endothelial proliferative mechanisms. Aim of this study therefore is to evaluate the therapeutic effects of PDT using animal PH model. Methods and Materials To induce PH, Sprague Dawley rats were injected with 60mg/kg monocrotaline (MCT). At day 7, PDT (630nm, 50 J/cm 2 ) using the photosensitizer, aminolevulinic acid (ALA) was administered. Hemodynamic measurements were taken on day 21 and lung and heart samples were dissected for molecular protein analysis. Results Rats treated with PDT using ALA had a significant reduction in right ventricular systolic pressure (RVSP), implying lower pulmonary arterial pressure, and a decreased ratio of RV/LV+S (right ventricle/left ventricle + septum), signifying reduced RV hypertrophy, compared with MCT-PH rats that received solely laser treatment. We observed a significant increase in P21Cip expression and decreased CKD1 and PCNA expression in the rat receiving PDT treatment. Hematoxylin and immunostaining showed PDT partially reverse MCT induced vascular remodeling in the lung treated versus untreated rats. Conclusions The clinical relevance of the findings in animal models will be assessed in human PAH lung tissue after lung transplantation. With these studies, we hope to provide new approaches for the treatment of this devastating disease.

Global Identification of Genes Related to Nutrient Deficiency in Intervertebral Disc Cells in an Experimental Nutrient Deprivation Model

BackgroundIntervertebral disc degeneration is a significant cause of degenerative spinal diseases. Nucleus pulposus (NP) cells reportedly fail to survive in large degenerated discs with limited nutrient availability. Therefore, understanding the regulatory mechanism of the molecular response of NP cells to nutrient deprivation may reveal a new strategy to treat disc degeneration. This study aimed to identify genes related to nutrient deprivation in NP cells on a global scale in an experimental nutrient deprivation model.Methodology/Principal FindingsRat NP cells were subjected to serum starvation. Global gene expression was profiled by microarray analysis. Confirmation of the selected genes was obtained by real-time polymerase chain reaction array analysis. Western blotting was used to confirm the expression of selected genes. Functional interactions between p21Cip1 and caspase 3 were examined. Finally, flow cytometric analyses of NP cells were performed. Microarray analysis revealed 2922 differentially expressed probe sets with ≥1.5-fold changes in expression. Serum starvation of NP cells significantly affected the expression of several genes involved in DNA damage checkpoints of the cell cycle, including Atm, Brca1, Cdc25, Gadd45, Hus1, Ppm1D, Rad 9, Tp53, and Cyclin D1. Both p27Kip1 and p53 protein expression was upregulated in serum-starved cells. p21Cip1 expression remained in NP cells transfected with short interfering RNA targeting caspase 3 (caspase 3 siRNA). Both G1 arrest and apoptosis induced by serum starvation were inhibited in cells transfected with caspase 3 siRNA.Conclusions/SignificanceNutrient deprivation in NP cells results in the activation of a signaling response including DNA damage checkpoint genes regulating the cell cycle. These results provide novel possibilities to improve the success of intervertebral disc regenerative techniques.

Protective Roles of Epithelial Cells in the Survival of Adult T-Cell Leukemia/Lymphoma Cells

Adult T-cell leukemia/lymphoma (ATL) is a highly invasive and intractable T-cell malignancy caused by human T-cell leukemia virus-1 infection. We demonstrate herein that normal tissue-derived epithelial cells (NECs) exert protective effects on the survival of leukemic cells, which may partially account for high resistance to antileukemic therapies in patients with ATL. Viral gene-silenced, ATL-derived cell lines (ATL cells) dramatically escaped from histone deacetylase inhibitor-induced apoptosis by direct co-culture with NECs. Adhesions to NECs suppressed p21(Cip1) expression and increased a proportion of resting G0/G1 phase cells in trichostatin A (TSA)-treated ATL cells. ATL cells adhering to NECs down-regulated CD25 expression and enhanced vimentin expression, suggesting that most ATL cells acquired a quiescent state by cell-cell interactions with NECs. ATL cells adhering to NECs displayed highly elevated expression of the cancer stem cell marker CD44. Blockade of CD44 signaling diminished the NEC-conferred resistance of ATL cells to TSA-induced apoptosis. Co-culture with NECs also suppressed the expression of NKG2D ligands on TSA-treated ATL cells, resulting in decreased natural killer cell-mediated cytotoxicity. Combined evidence suggests that interactions with normal epithelial cells augment the resistance of ATL cells to TSA-induced apoptosis and facilitate immune evasion by ATL cells.

Endometrial stromal sarcoma: clinicopathological and immunophenotypic study of 16 cases

Endometrial stromal sarcomas (ESSs) are rare tumors and are divided into two groups: low-grade endometrial stromal sarcoma (ESS-LG) and undifferentiated endometrial sarcoma (UES). The purpose of this study was to compare the clinicopathological features and immunophenotypes of ESS-LG and UES. The authors evaluated 16 patients diagnosed with ESS at the Hyogo Cancer Center, reviewed their files and data, and performed an immunohistochemical study for oncogenic proteins (EGFR, PDGFR-α, and PDGFR-β) and cell cycle regulators (cyclin D1, cyclin E, p16(INK4a), p21(cip1), p27(kip1), and p53) to compare ESS-LG and UES using the World Health Organization (WHO) classification. Four cases (25 %) were classified as ESS-LGs and 12 (75 %) as UES. Patients with UES had a significantly worse overall survival than did those with ESS-LG (p = 0.0445). Although no ESS-LGs showed expression of p16(INK4a), 10 of 12 (83 %) UESs showed expression of p16(INK4a). UESs showed a trend toward higher expression of cyclin D1, p21(cip1), and p53 compared with ESS-LGs. Our data emphasize the clinical importance of the WHO classification of ESS. It is of utmost importance to establish a proper classification to increase the consistency of data that may be useful for improving clinical and therapeutic management of patients with ESS.

Changes in Tumor Morphology and Cyclin-Dependent Kinase Inhibitor Expression in Metastatic Melanoma Treated With Selective Second-Generation BRAF Inhibitor

Dermatologic toxicities associated with anticancer-targeted therapy include hand-foot skin reactions, vasculitis, cutaneous epithelial proliferations, such as keratosis, keratoacanthoma, and invasive squamous cell carcinoma. In this case report, we describe alterations of tumor morphology and patterns of cyclin-dependent kinase inhibitor (CDKI) expression in a patient who received GSK2118436, a second-generation RAF inhibitor, for stage IV (M1c) metastatic melanoma. To explore the effects of GSK2118436 on the expression patterns of CDKI (p16, p21, p27, p57), we immunohistochemically evaluated in vivo melanoma cells pre- and posttreated with GSK2118436. After GSK2118436 treatment, the melanoma cells decreased in size and demonstrated hyperchromatic nuclei and indistinct nucleoli. p16 was strongly expressed in the cytoplasm of pretreated melanoma cells and in the nucleus and cytoplasm in posttreated melanoma cells. Expression of both p27 (nucleus) and p57 (cytoplasm) was increased in posttreated melanoma cells and no significant difference in p21 expression was noted in either pre- or posttreated tumor cells. These findings may help explain the molecular mechanisms by which tumor cells retain the ability to proliferate. The persistent activation of pro-oncogenic activities of CDKI (eg, p27 and p57) and/or compartmentalization of p16 in cytoplasm or nucleus may allow tumor cells to bypass BRAF-induced senescence mechanisms. Furthermore, awareness of changes in tumor morphology after treatment with RAF inhibitors may be helpful in histologic evaluation of the spectrum of dermatologic toxicity, which may occur on targeted therapy.

Homocysteine Inhibits Hepatocyte Proliferation via Endoplasmic Reticulum Stress

Homocysteine is an independent risk factor for coronary, cerebral, and peripheral vascular diseases. Recent studies have shown that levels of homocysteine are elevated in patients with impaired hepatic function, but the precise role of homocysteine in the development of hepatic dysfunction is unclear. In this study, we examined the effect of homocysteine on hepatocyte proliferation in vitro. Our results demonstrated that homocysteine inhibited hepatocyte proliferation by up-regulating protein levels of p53 as well as mRNA and protein levels of p21Cip1 in primary cultured hepatocytes. Homocysteine induced cell growth arrest in p53-positive hepatocarcinoma cell line HepG2, but not in p53-null hepatocarcinoma cell line Hep3B. A p53 inhibitor pifithrin-α inhibited the expression of p21Cip1 and attenuated homocysteine-induced cell growth arrest. Homocysteine induced TRB3 expression via endoplasmic reticulum stress pathway, resulting in Akt dephosphorylation. Knock-down of endogenous TRB3 significantly suppressed the inhibitory effect of homocysteine on cell proliferation and the phosphorylation of Akt. LiCl reversed homocysteine-mediated cell growth arrest by inhibiting TRB3-mediated Akt dephosphorylation. These results demonstrate that both TRB3 and p21Cip1 are critical molecules in the homocysteine signaling cascade and provide a mechanistic explanation for impairment of liver regeneration in hyperhomocysteinemia.

Influence of Block of NF-Kappa B Signaling Pathway on Oxidative Stress in the Liver Homogenates

The aim of the present study was to assess whether BAY 11-7082, a nuclear factor-kappaB (NF-κB) inhibitor, influences the level of reactive oxygen species (ROS), tumor necrosis factor alpha (TNF-α), and NF-κB related signaling pathways in the liver. The animals were divided into 4 groups: I: saline; II: saline + endothelin-1 (ET-1) (1.25 μg/kg b.w., i.v.); III: saline + ET-1 (12.5 μg/kg b.w., i.v.); and IV: BAY 11-7082 (10 mg/kg b.w., i.v.) + ET-1 (12.5 μg/kg b.w., i.v.). Injection of ET-1 alone at a dose of 12.5 μg/kg b.w. showed a significant (P < 0.001) increase in thiobarbituric acid reactive substances (TBARS) and hydrogen peroxide (H2O2) level and decrease (P < 0.01) in GSH level (vs. control). ET-1 administration slightly downregulated gene expression of p65 of NF-κB but potently and in a dose-dependent way downregulated p21-cip gene expression in the liver. BAY 11-7082 significantly decreased TBARS (P < 0.001), H2O2 (P < 0.01) and improved the redox status (P < 0.05), compared to ET-1 group. The concentration of TNF-α was increased in the presence of ET-1 (P < 0.05), while BAY 11-7082 decreased TNF-α concentration (P < 0.01). Inhibition of IkBα before ET-1 administration downregulated gene expression of p21-cip but had no effect on p65.

HDAC Inhibitor L-Carnitine and Proteasome Inhibitor Bortezomib Synergistically Exert Anti-Tumor Activity In Vitro and In Vivo

Combinations of proteasome inhibitors and histone deacetylases (HDAC) inhibitors appear to be the most potent to produce synergistic cytotoxicity in preclinical trials. We have recently confirmed that L-carnitine (LC) is an endogenous HDAC inhibitor. In the current study, the anti-tumor effect of LC plus proteasome inhibitor bortezomib (velcade, Vel) was investigated both in cultured hepatoma cancer cells and in Balb/c mice bearing HepG2 tumor. Cell death and cell viability were assayed by flow cytometry and MTS, respectively. Gene, mRNA expression and protein levels were detected by gene microarray, quantitative real-time PCR and Western blot, respectively. The effect of Vel on the acetylation of histone H3 associated with the p21cip1 gene promoter was examined by using ChIP assay and proteasome peptidase activity was detected by cell-based chymotrypsin-like (CT-like) activity assay. Here we report that (i) the combination of LC and Vel synergistically induces cytotoxicity in vitro; (ii) the combination also synergistically inhibits tumor growth in vivo; (iii) two major pathways are involved in the synergistical effects of the combinational treatment: increased p21cip1 expression and histone acetylation in vitro and in vivo and enhanced Vel-induced proteasome inhibition by LC. The synergistic effect of LC and Vel in cancer therapy should have great potential in the future clinical trials.

Dexamethasone Reduces Sensitivity to Cisplatin by Blunting p53-Dependent Cellular Senescence in Non-Small Cell Lung Cancer

IntroductionDexamethasone (DEX) co-treatment has proved beneficial in NSCLC patients, improving clinical symptoms by the reduction of side effects after chemotherapy. However, recent studies have shown that DEX could render cancer cells more insensitive to cytotoxic drug therapy, but it is not known whether DEX co-treatment could influence therapy-induced senescence (TIS), and unknown whether it is in a p53-dependent or p53-independent manner.MethodsWe examined in different human NSCLC cell lines and detected cellular senescence after cisplatin (DDP) treatment in the presence or absence of DEX. The in vivo effect of the combination of DEX and DDP was assessed by tumor growth experiments using human lung cancer cell lines growing as xenograft tumors in nude mice.ResultsCo-treatment with DEX during chemotherapy in NSCLC resulted in increased tumor cell viability and inhibition of TIS compared with DDP treated group. DEX co-treatment cells exhibited the decrease of DNA damage signaling pathway proteins, the lower expression of p53 and p21CIP1, the lower cellular secretory program and down-regulation of NF-κB and its signaling cascade. DEX also significantly reduced DDP sensitivity in vivo.ConclusionsOur results underscore that DEX reduces chemotherapy sensitivity by blunting therapy induced cellular senescence after chemotherapy in NSCLC, which may, at least in part, in a p53-dependent manner. These data therefore raise concerns about the widespread combined use of gluocorticoids (GCs) with antineoplastic drugs in the clinical management of cancer patients.

Progestin drives breast cancer growth by inducing p21CIP1 expression through the assembly of a transcriptional complex among Stat3, progesterone receptor and ErbB-2

Cell cycle regulator p21CIP1 has controversial biological effects in breast cancer since in spite of its role as cell cycle inhibitor and promoter of cellular senescence, it also induces cell proliferation and chemoteraphy resistance. We here explored the molecular mechanisms involved in progestin regulation of p21CIP1 expression. We also investigated the biological effects of p21CIP1 in breast cancer cells. We found that the synthetic progestin medroxyprogesterone acetate (MPA) upregulates p21CIP1 protein expression via c-Src, signal transducer and activator of transcription 3 (Stat3) and ErbB-2 phosphorylation. Notably, we also found that ErbB-2 nuclear function plays a key role in MPA-induction of p21CIP1 expression. Interestingly, we determined that progestin drives p21CIP1 transcriptional activation via a novel nonclassical transcriptional mechanism in which progesterone receptor is recruited along with Stat3 and ErbB-2 to a Stat3 binding site at p21CIP1 promoter. Our findings revealed that ErbB-2 functions as a coactivator of Stat3 in progestin induction of p21CIP1 transcriptional activation. Furthermore, we demonstrated that blockage of p21CIP1 expression strongly inhibited in vitro and in vivo progestin-induced breast cancer cell proliferation. These results further support the hypothesis that according to cell context and type of stimulus, p21CIP1 is capable of inducing cell cycle progression. Moreover, we provided evidence that Stat3 and nuclear ErbB-2 are key players in progestin-induced p21CIP1 regulation.

Cross-Species Investigations Reveal Role, Mechanism and Predictive Power of Paracrine, Secondary Senescence in B-Cell Lymphoma Therapy

AbstractAbstract 3715Apoptosis and cellular senescence operate as anti-tumor safeguard mechanisms. Unlike apoptotic cells, senescent cells remain viable, and, hence, may crosstalk to other cells in their vicinity over extended periods of time. In fact, cells that entered oncogene-induced senescence or anticancer therapy-induced senescence (TIS) present with a senescence-associated secretory phenotype (SASP), a massive production of secretable factors, which reportedly reinforces senescence through an intracellular mechanism. Utilizing the Eμ-myc transgenic mouse lymphoma model, we provide evidence for an outcome-relevant paracrine, DNA damage-independent secondary senescence program (SecS) in vitro and in vivo.Apoptosis-blocked (bcl2-infected) lymphoma cells from different genetic backgrounds were treated with the DNA-damaging anticancer agent adriamycin in vitro or the alkylating agent cyclophosphamide upon lymphoma formation in mice in vivo. TIS and SecS was detected based on senescence-associated b-galactosidase activity (SA-b-gal), Ki67 staining and BrdU incorporation. The secretome of senescent cells was analyzed by proteomics, gene expression and protein arrays. Overall and progression free survival in mice and patients was assessed by Kaplan-Meier analysis.Transcriptome and secretome analyses followed by functional studies found extracellular matrix proteins, especially small leucine-rich proteoglycans (SLRP), but not NF-kB-dependent cytokines and chemokines, to induce SecS in proliferating lymphoma cells in a paracrine fashion, and linked a “high secretor” status to stronger SecS induction. Dissecting senescence-mediating pathways in recipient cells by biochemical, genetic and pharmacological means unveiled an essential role for the LDL receptor-related protein 1 (LRP1), a receptor for SLRP and other SASP components, through the cell-cycle inhibitor p21CIP1 in SecS. Accordingly, mice harboring TIS-capable but genetically SecS-defective lymphomas (e.g. lacking LRP1 or p21CIP1 expression) experienced inferior long-term outcome to therapy. Not only the recipient cell-based LRP1 status but also the genetically and biologically distinct donor cell-based secretor gene signature stratified outcome in mice. Strikingly, humanized versions of both classifiers were predictive in a large cohort of diffuse large B-cell lymphoma (DLBCL) patients, where they identified – although composed of different gene sets – largely overlapping patient subgroups with superior prognosis, again suggesting SecS as the critical underlying treatment effector principle. Our study highlights the predictive power of senescence for treatment outcome in DLBCL, and provides functional examples (which will be discussed at the meeting) for SASP-related non-genotoxic pro-senescent therapies. Disclosures:No relevant conflicts of interest to declare.

Targeting the Hedgehog Signaling Pathway Limits the Self-Renewal of BCR-ABL1 Positive Leukemia Cells: Molecular Mechanisms

Abstract 531Hematopoietic stem cells and leukemic stem cells share common features, including self-renewal, the capacity to differentiate, resistance to apoptosis, and limitless proliferative potential. Despite these similarities, several stemness factors, such as Hedgehog, Wnt, Notch, and BMI-1 show differential activation in normal versus leukemia stem cells. Hedgehog signaling is increased in BCR-ABL1 positive stem and progenitor cells becoming more active with disease progression. We have previously shown that the combination with the hedgehog signaling pathway inhibitor, vismodegib and ABL tyrosine kinase inhibitor (ABL TKIs) inhibits the minimal residual cells in BCR-ABL1-positive leukemia cells (Blood: ASH Annual Abstracts, Nov 2011; 118: 63). In the present study, we investigated the molecular mechanisms by which vismodegib and LDE225 regulate the self-renewal of primary BCR-ABL1 positive leukemia cells in vivo. To identify the leukemia-propagating cell fraction of BCR-ABL1-positive leukemia, we serially transplanted human leukemia cells from patients with chronic myeloid leukemia blast crisis (n=1; T315I BCR-ABL1) or Ph-positive acute lymphoblastic leukemia (n=2, T315I BCR-ABL1 and WT-BCR-ABL1) into NOD/SCID/IL-2γc−/− mice. The cell fractions with CD34+CD38−CD19+and CD34+CD38+CD19+ could self-renew and transfer the leukemia in NOD/SCID mice. To investigate the effects of hedgehog inhibition on self-renewal and the relevance of the hedgehog pathway as a therapeutic target in BCR-ABL1 positive leukemia, we examined the activity of vismodegib and LDE225 against CD34+CD38−CD19+, CD34+CD38+CD19+ fractions transferred NOD/SCID mice in vivo. NOD/SCID mice were injected intravenously with BCR-ABL1 positive cells then treated with vismodegib (20 mg/kg; p.o.) or LDE225 (20 mg/kg; p.o.) for 28 days. All mice demonstrated the engraftment of leukemia by flow cytometry. However, the treatment with vismodegib or LDE225 reduced the population of CD34+CD38− positive cells. We isolated human CD45+ cells from the spleen of mice from each treatment group and injected equivalent numbers of leukemia cells into secondary recipients, subsequently treated with vismodegib or LDE225 for 28 days. Following 30 days, all mice received BCR-ABL1 cells from vehicle treated mice engrafted with leukemia. In contrast, leukemia engraftment was not detected in recipient mice (n=6) from vismodegib or LDE225 treated donors. These results demonstrate the persistent effects of hedgehog inhibition on long term self-renewing BCR-ABL1-positive leukemia cells. We further examined the effects of hedgehog pathway modulation on in vitro clonogenic growth. CD34+CD38−CD19+ cells from T315I BCR-ABL1 (n=2) and WT-BCR-ABL1 (n=1) cells were treated with 1 μM of vismodegib or 200 nM of LDE225 for 72 hrs, washed free of drugs, and plated in quadruplicate in methylcellulose. At 14 days, colonies were counted as initial plating. The representative plate was then washed and cells were re-suspended and re-plated. After an additional 14 days, colonies were counted as secondary re-plating. Clonogenic recovery of untreated cells was normalized to 100% and plating results from all treatment groups were expressed as % control. Hedgehog pathway inhibition by vismodegib and LDE225 had only minimum effects on colony formation after initial plating over control cells. However, upon serial re-plating, secondary colony formations were significantly inhibited by vismodegib and LDE225 (p<0.001). To identify the mechanisms that limit the self-renewal of BCR-ABL1-positive cells by vismodegib and LDE225, NOD/SCID mice engrafted with WT-BCR-ABL1-positive CD34+ CD19+ fractions were treated with vismodegib (20 mg/kg; p.o.) or LDE225 (20 mg/kg; p.o.) for 14 days. Both vismodegib and LDE225 induced the expressions of p21Cip1, pATM, pChk2 and γH2AX related with DNA damage response, and reduced the expression of Gli-1, Gli-2, Bcl-2, and cyclin D2. Our preclinical results indicate that vismodegib and LDE225 have potential as an important option for controlling the drug-resistant leukemia initiating cells in BCR-ABL1 positive leukemia. Although several hedgehog inhibitors have now entered clinical evaluation, it is expected that hedgehog inhibitors may become extremely useful therapeutic interventions in a number of hematological neoplasms, including BCR-ABL1 positive leukemia, where the persistence of cancer stem cells. Disclosures:No relevant conflicts of interest to declare.

Curcumin enhances the response of non-Hodgkin’s lymphoma cells to ionizing radiation through further induction of cell cycle arrest at the G2/M phase and inhibition of mTOR phosphorylation

It is crucial to enhance tumor radiosensitivity for the purpose of both lowering the dose of ionizing radiation (IR) and achieving higher antitumor efficacy. We identified curcumin as a radiosensitizer to enhance non-Hodgkin's lymphoma (NHL) cell response to IR in vitro and further investigated the mechanism mediating this effect. We treated Namalwa, Ramos and Raji cell lines with vehicle, curcumin, IR and curcumin-IR. Cell viability and cell cycle distribution were determined to ascertain the radiosensitization effect of curcumin. DNA damage-related proteins, cell cycle regulatory proteins, phosphorylation of mammalian target of rapamycin (mTOR) and the nuclear translocation of the downstream nuclear factor-κB (NF-κB) target were examined by western blotting. Treatment with curcumin led to decreased viability of all three types of NHL cells and had a profound radiosensitization effect. Pre-treatment with curcumin at a low concentration of 2 µmol/l increased IR-induced G2/M arrest in the cell cycle and increased the expression of cyclin-dependent kinase inhibitors, p21cip1 and p53. However, this effect was blocked when NHL cells were pre-treated with 10 µmol/l of KU55933, a specific inhibitor of ataxia-telangiectasia-mutated (ATM). Pre-treatment with curcumin inhibited the phosphorylation of mTOR and the nuclear translocation of the downstream NF-κB target induced by IR. Curcumin enhanced the cell response to IR in NHL mediated through the induction of G2/M phase arrest and the inhibition of both a constitutive and IR-induced activation of the mTOR-NF-κB pathway. This offers great potential for curcumin to be used in conjunction with radiotherapy for NHL in order to increase the efficiency of the treatment.

Upregulation of miR‐153 promotes cell proliferation via downregulation of the PTEN tumor suppressor gene in human prostate cancer

Accumulating evidence indicates that microRNAs play a pivotal role in the development and progression of prostate cancer. The present study was aimed at clarifying the biological functions of miR-153, one of the upregulated microRNAs in prostate cancers, and the signaling transduction induced by miR-153. miR-153 was identified to be overexpressed in prostate cancers. The probable biological function of miR-153 in cellular proliferation was then examined by diverse assays, such as MTT, colony formation and BrdUrd incorporation assay. Moreover, real-time PCR and western blotting analysis were applied to investigate the underlying molecular mechanism induced by miR-153. Luciferase assays were used to determined the FOXO1 transactivity and the direct regulation of PTEN-3'-UTR by miR-153. High-throughput method identified miR-153 to be upregulated in prostate cancers, which is further confirmed by the upregulated expression in four paired prostate tumor/adjacent non-cancerous tissues from the same patients. Further studies revealed that overexpression of miR-153 promoted cell cycle transition and cell proliferation, while inhibition of miR-153 reduced this effect. Moreover, miR-153 overexpression in prostate cancer cells increased the G1/S transitional promoter, cyclin D1 expression, and decreased cyclin-dependent kinase (CDK) inhibitor, p21(Cip1) expression. In addition, we demonstrated that miR-153 directly targeted the PTEN tumor suppressor gene, activated the AKT signaling and downregulated FOXO1 transcriptional activity. Taken together, our results suggest that miR-153 play an important role in promoting proliferation of human prostate cancer cells and present a novel mechanism of microRNA-mediated direct suppression of PTEN expression in prostate cancer cells.

Comparison of Gene Expression Profiling in Sarcomas and Mesenchymal Stem Cells Identifies Tumorigenic Pathways in Chemically Induced Rat Sarcoma Model

Mesenchymal stem cells (MSCs) are believed to be the cell of origin for most sarcomas including osteosarcoma and malignant fibrous histiocytoma (MFH/UPS). To identify the signaling pathways involved in sarcoma pathogenesis, we compared gene expression profiles in rat osteosarcoma and MFH cells with those in syngeneic rat MSCs. Analysis of genes that characterize MSCs such as CD44, CD105, CD73, and CD90 showed higher expression in MSCs compared to sarcomas. Pathways involved in focal and cell adhesion, cytokine-cytokine receptors, extracellular matrix receptors, chemokines, and Wnt signaling were down-regulated in both sarcomas. Meanwhile, DNA replication, cell cycle, mismatch repair, Hedgehog signaling, and metabolic pathways were upregulated in both sarcomas. Downregulation of p21Cip1 and higher expression of CDK4-cyclinD1 and CDK2-cyclinE could accelerate cell cycle in sarcomas. The current study indicated that these rat sarcomas could be a good model for their human counterparts and will provide the further insights into the molecular pathways and mechanisms involved in sarcoma pathogenesis.

Activity of the selective IκB kinase inhibitor BMS-345541 against T-cell acute lymphoblastic leukemia

Several lines of evidence suggest that the IκB kinase (IKK)/nuclear factor-κB (NFκB) axis is required for viability of leukemic cells and is a predictor of relapse in T-cell acute lymphoblastic leukemia (T-ALL). Moreover, many anticancer agents induce NFκB nuclear translocation and activation of its target genes, which counteract cellular resistance to chemotherapeutic drugs. Therefore, the design and the study of IKK-specific drugs is crucial to inhibit tumor cell proliferation and to prevent cancer drug-resistance. Here, we report the anti-proliferative effects induced by BMS-345541 (a highly selective IKK inhibitor) in three Notch1-mutated T-ALL cell lines and in T-ALL primary cells from pediatric patients. BMS-345541 induced apoptosis and an accumulation of cells in the G2/M phase of the cell cycle via inhibition of IKK/NFκB signaling. We also report that T-ALL cells treated with BMS-345541 displayed nuclear translocation of FOXO3a and restoration of its functions, including control of p21Cip1 expression levels. We demonstrated that FOXO3a subcellular re-distribution is independent of AKT and ERK 1/2 signaling, speculating that in T-ALL the loss of FOXO3a tumor suppressor function could be due to deregulation of IKK, as has been previously demonstrated in other cancer types. It is well known that, differently from p53, FOXO3a mutations have not yet been found in human tumors, which makes therapeutics activating FOXO3a more appealing than others. For these features, BMS-345541 could be used alone or in combination with traditional therapies in the treatment of T-ALL.