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Water Extract of Ashwagandha Leaves Has Anticancer Activity: Identification of an Active Component and Its Mechanism of Action

BackgroundCancer is a leading cause of death accounting for 15-20% of global mortality. Although advancements in diagnostic and therapeutic technologies have improved cancer survival statistics, 75% of the world population live in underdeveloped regions and have poor access to the advanced medical remedies. Natural therapies hence become an alternative choice of treatment. Ashwagandha, a tropical herb used in Indian Ayurvedic medicine, has a long history of its health promoting and therapeutic effects. In the present study, we have investigated an anticancer activity in the water extract of Ashwagandha leaves (ASH-WEX).Methodology/Principal FindingsAnticancer activity in the water extract of Ashwagandha leaves (ASH-WEX) was detected by in vitro and in vivo assays. Bioactivity-based size fractionation and NMR analysis were performed to identify the active anticancer component(s). Mechanism of anticancer activity in the extract and its purified component was investigated by biochemical assays. We report that the ASH-WEX is cytotoxic to cancer cells selectively, and causes tumor suppression in vivo. Its active anticancer component was identified as triethylene glycol (TEG). Molecular analysis revealed activation of tumor suppressor proteins p53 and pRB by ASH-WEX and TEG in cancer cells. In contrast to the hypophosphorylation of pRB, decrease in cyclin B1 and increase in cyclin D1 in ASH-WEX and TEG-treated cancer cells (undergoing growth arrest), normal cells showed increase in pRB phosphorylation and cyclin B1, and decrease in cyclin D1 (signifying their cell cycle progression). We also found that the MMP-3 and MMP-9 that regulate metastasis were down regulated in ASH-WEX and TEG-treated cancer cells; normal cells remained unaffected.ConclusionWe provide the first molecular evidence that the ASH-WEX and TEG have selective cancer cell growth arrest activity and hence may offer natural and economic resources for anticancer medicine.

Targeted disruption of the EZH2–EED complex inhibits EZH2-dependent cancer

Enhancer of zeste homolog2 (EZH2) is the histone lysine N-methyltransferase component of the Polycomb repressive complex 2 (PRC2), which in conjunction with embryonic ectoderm development (EED) and suppressor of zeste 12 homolog (SUZ12), regulates cell lineage determination and homeostasis. Enzymatic hyperactivity has been linked to aberrant repression of tumor suppressor genes in diverse cancers. Here, we report the development of stabilized alpha-helix of EZH2 (SAH-EZH2) peptides that selectively inhibit H3 Lys27 trimethylation by dose-responsively disrupting the EZH2/EED complex and reducing EZH2 protein levels, a mechanism distinct from that reported for small molecule EZH2 inhibitors targeting the enzyme catalytic domain. MLL-AF9 leukemia cells, which are dependent on PRC2, undergo growth arrest and monocyte/macrophage differentiation upon treatment with SAH-EZH2, consistent with observed changes in expression of PRC2-regulated, lineage-specific marker genes. Thus, by dissociating the EZH2/EED complex, we pharmacologically modulate an epigenetic “writer” and suppress PRC2-dependent cancer cell growth.

678 The Bone Morphogenetic Protein Pathway Either Enhances or Inhibits the Wnt Pathway Depending on SMAD4 and p53 Status in Colorectal Cancer

Introduction:Notch1-mediated signaling influences cell fate decisions, cell cycle and carcinogenesis via a transcription activation complex consisting of ICN1 (the activated form of Notch1), the co-activatorMAML and the transcription factor CSL. However, the consequences of Notch activation are complex and context dependent. The role of Notch signaling in esophageal epithelial biology andmalignant transformation remain elusive. Methods:Human esophageal keratinocytes immortalized by telomerase (EPC1-hTERT and EPC2-hTERT) as well as non-transformed and transformed derivatives including EPC2T overexpressing EGFR, cyclin D1 and p53R175H; transformed human esophageal keratinocytes carrying a human papilloma virus (HPV)-E6E7 fusion oncogene (EN60) and esophageal squamous cell carcinoma (ESCC) cell lines were genetically engineered to express tetracycline-inducible ICN1; dominant-negative MAML1 (DNMAML1), a genetic pan-Notch inhibitor; or short hairpin RNA (shRNA) directed against CSL, HPV-E7 or cyclin-dependent kinase inhibitor p16INK4A. Notch activity was determined by transfection assays of an 8xCSL-luciferase reporter construct. Notch target genes were determined by quantitative RT-PCR. Western blotting determined retinoblastoma (RB) protein phosphorylation status. Cell proliferation was determined by colorimetric assays. Anchorage-independent colony formation was evaluated in soft agar. Tumor formation was assessed by xenograft transplantation in immunodeficient mice. Senescence associated β-galactosidase (SABG) activity was determined. Results: ICN1 activated CSL-dependent transcription in all cell lines tested and this activation was antagonized by either DNMAML1 expression or CSL knockdown. In response to ICN1 induction, nontransformed cells underwent growth arrest displaying flat and enlarged cell morphology with SABG positive staining (40-80%), suggesting senescence. ICN1-mediated senescence was found to be CSL-dependent and was associated with decreased RB protein phosphorylation and p16INK4A upregulation. ICN1-mediated senescence was inhibited by p16INK4A knockdown, but not the presence of p53R175H. ICN1 failed to induce senescence in EN60 cells expressing EPV-E7 protein or ESCC cell lines lacking the INK4A locus. ICN1 stimulated colony formation and tumor growth in these transformed cell lines. HPV E7 knockdown in EN60 cells resulted in senescence with activation of endogenous Notch1. Conclusions: These novel data indicate that loss of the RB protein-dependent senescence checkpoint function may be permissive for Notch1 to facilitate malignant transformation in human esophageal epithelial cells.

677 Inactivation of the Retinoblastoma Protein-Dependent Senescence Checkpoint Functions Permits Notch1 to Gain Oncogenic Tumor Promoting Activity

Introduction:Notch1-mediated signaling influences cell fate decisions, cell cycle and carcinogenesis via a transcription activation complex consisting of ICN1 (the activated form of Notch1), the co-activatorMAML and the transcription factor CSL. However, the consequences of Notch activation are complex and context dependent. The role of Notch signaling in esophageal epithelial biology andmalignant transformation remain elusive. Methods:Human esophageal keratinocytes immortalized by telomerase (EPC1-hTERT and EPC2-hTERT) as well as non-transformed and transformed derivatives including EPC2T overexpressing EGFR, cyclin D1 and p53R175H; transformed human esophageal keratinocytes carrying a human papilloma virus (HPV)-E6E7 fusion oncogene (EN60) and esophageal squamous cell carcinoma (ESCC) cell lines were genetically engineered to express tetracycline-inducible ICN1; dominant-negative MAML1 (DNMAML1), a genetic pan-Notch inhibitor; or short hairpin RNA (shRNA) directed against CSL, HPV-E7 or cyclin-dependent kinase inhibitor p16INK4A. Notch activity was determined by transfection assays of an 8xCSL-luciferase reporter construct. Notch target genes were determined by quantitative RT-PCR. Western blotting determined retinoblastoma (RB) protein phosphorylation status. Cell proliferation was determined by colorimetric assays. Anchorage-independent colony formation was evaluated in soft agar. Tumor formation was assessed by xenograft transplantation in immunodeficient mice. Senescence associated β-galactosidase (SABG) activity was determined. Results: ICN1 activated CSL-dependent transcription in all cell lines tested and this activation was antagonized by either DNMAML1 expression or CSL knockdown. In response to ICN1 induction, nontransformed cells underwent growth arrest displaying flat and enlarged cell morphology with SABG positive staining (40-80%), suggesting senescence. ICN1-mediated senescence was found to be CSL-dependent and was associated with decreased RB protein phosphorylation and p16INK4A upregulation. ICN1-mediated senescence was inhibited by p16INK4A knockdown, but not the presence of p53R175H. ICN1 failed to induce senescence in EN60 cells expressing EPV-E7 protein or ESCC cell lines lacking the INK4A locus. ICN1 stimulated colony formation and tumor growth in these transformed cell lines. HPV E7 knockdown in EN60 cells resulted in senescence with activation of endogenous Notch1. Conclusions: These novel data indicate that loss of the RB protein-dependent senescence checkpoint function may be permissive for Notch1 to facilitate malignant transformation in human esophageal epithelial cells.

Abstract 2321: Rapid induction of apoptosis by PI3K inhibitors is dependent upon their transient inhibition of RAS signaling.

Abstract Hyperactivation of the PI3K/AKT/mTOR pathway in tumors causes feedback inhibition throughout the signaling network, including inhibition of receptor tyrosine kinase (RTK) expression and function. Exposure of these tumors to mTOR or AKT inhibitors relieves this feedback and reactivates RTK signaling. RTK reactivation promotes PI3K activity and attenuates the antitumor effects of the drugs. We have now characterized the details of PI3K-induced feedback using a selective class 1 PI3K inhibitor and compared its effects with those elicited by a selective allosteric inhibitor of AKT. In breast cancer cell lines with dysregulation of PI3K signaling, both drugs cause marked inhibition of AKT phosphorylation and relief of feedback inhibition of HER3, IGF1, and insulin RTKs. Whereas both inhibitors cause tumor cells to undergo growth arrest, only the PI3K inhibitor causes a rapid induction of apoptosis within 2 hours of drug addition. As opposed to the PI3K inhibitor, the AKT inhibitor induces PI3K activity and a set of substrates that are inhibited by the PI3K inhibitor. We therefore sought to identify processes that are selectively inhibited by the PI3K inhibitor to explain its selective induction of apoptosis. AKT inhibition causes the induction of ERK phosphorylation in these cells. In contrast, we found that PI3K inhibition causes rapid inhibition of RAS-GTP levels, RAF activation and ERK phosphorylation in tumor cells with wild type RAS, but not those with mutant RAS. Inhibition of RAS is transient, recovering 2-3 hours after drug addition. We determined that adding MEK inhibitors to AKT inhibitors is similarly associated with a rapid induction of apoptosis within 2 hours. Moreover, addition of EGF to tumor cells co-treated with PI3K inhibitors prevents ERK inhibition and apoptosis, whereas addition of MEK inhibitors to the PI3K inhibitor, EGF treated cells restores the apoptotic phenotype. We conclude that PI3K is upstream of both RAS and AKT in RAS wild type tumors. Inhibitors of PI3K cause sustained inhibition of AKT and transient inhibition of RAS with both effects required for rapid induction of apoptosis. These data suggest that pulsatile inhibition of PI3K may maximize the therapeutic efficacy of this approach. Citation Format: Marie Will, Weiyi Toy, Zhan Yao, Poulikos Poulikakos, Vanessa Rodrik-Outmezguine, Claudia Schneider, Juliane Paul, Jose Baselga, Ningshu Liu, Neal Rosen, Sarat Chandarlapaty. Rapid induction of apoptosis by PI3K inhibitors is dependent upon their transient inhibition of RAS signaling. [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 2321. doi:10.1158/1538-7445.AM2013-2321

Abstract SY15-01: Merkel cell polyomavirus: its role in pathogenesis and its potential as a molecular target.

Abstract Merkel cell carcinoma (MCC) was first described as a pathologic entity in 1972, and is an uncommon but aggressive skin cancer with a high propensity for metastasis and recurrence. In 2008, a new human polyomavirus, Merkel cell polyomavirus (MCV) was discovered by digital transcriptome subtraction in an MCC lesion and is now one of eight new human polyomaviruses described in the past five years. MCV is part of the normal, healthy skin flora but causes cancer after viral genome mutations eliminate its replication capacity. Further, MCV is clonally integrated in 70-80% of MCC tumors suggesting a primary role for its involvement in MCV infected MCCs. The MCV genome is ~5400bp and has features including early, late, and non-coding regulatory regions conserved with other polyomaviruses. The MCV early region encodes for two potential viral oncoproteins: large T antigen (LT) and small T antigen (ST). Experiments show these proteins are expressed in MCC tumor cells from patient biopsies, and that MCV-positive MCC cell lines undergo growth arrest and/or cell death upon T antigen knockdown by short hairpin RNA (shRNA). These experiments provide direct experimental evidence that T antigen expression is necessary for the maintenance of MCV-positive MCC and that MCV is the infectious cause of MCV-positive MCC. While similar to known polyomaviruses, MCV oncogenes act in new ways, such as activation of the survivin oncoprotein and PP2A-independent targeting of cap-dependent translation. In five years, the diagnosis and treatment potential for an intractable and enigmatic cancer has dramatically changed through the discovery of a viral cause of MCC. Citation Format: Yuan Chang. Merkel cell polyomavirus: its role in pathogenesis and its potential as a molecular target. [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 SY15-01. doi:10.1158/1538-7445.AM2013-SY15-01

Mimicking p14ARF Phosphorylation Influences Its Ability to Restrain Cell Proliferation

The INK4a/ARF locus on the short arm of chromosome 9 is one of the most frequently altered loci in human cancer. It is generally accepted that ARF is involved in oncogenic checkpoint pathways by sensitizing incipient cancer cells to undergo growth arrest or apoptosis through both p53-dependent and independent pathways. While intensive studies have been focused on ARF activation at the transcriptional level, only recently mechanisms governing ARF turnover have been identified. Here, we show for the first time that p14ARF is a PKC target. Prediction analysis showed many potential phosphorylation sites in PKC consensus sequences within ARF protein, and, among them, the threonine at position 8 was the most conserved. Substitution of this threonine influences both ARF stability and localization. Furthermore, a phosphomimetic ARF mutation reduces the ability to arrest cell growth although the ability to bind MDM2 and stabilize p53 result unaffected. Thus we propose that phosphorylation of ARF in both immortalized and tumor cell lines could be a mechanism to escape ARF surveillance following proliferative and oncogenic stress.

Role of STAT3 and Th17 Cells in Cutaneous T Cell Lymphoma

AbstractAbstract 66 BackgroundCutaneous T cell Lymphoma (CTCL) is a heterogeneous group of non-Hodgkin's lymphomas characterized by skin-homing malignant T cells. The etiology of CTCL remains enigmatic, but constitutive Signal Transducer and Activator of Transcription 3 (STAT3) activity is characteristic of this malignancy. CTCL cell lines undergo growth arrest and apoptosis following inhibition of STAT3 signaling, suggesting that STAT3 may be a pro-survival factor in these cells. STAT3 phosphorylation is also required for the initiation and maintenance of the Th17 differentiation program, a recently identified subset of CD4+ T helper cells, implicated in a number of chronic inflammatory conditions including rheumatoid arthritis and psoriasis. Furthermore, we know that Histone Deacetylase inhibitors (HDACi) namely Vorinostat and Romidepsin are very active agents for treatment of CTCL but their mechanism of action is still to be elucidated. Our goal is to probe the contribution of Th17 mediated inflammation in CTCL and to examine whether HDAC inhibitors work by modulating Th17 inflammation in the context of this malignancy. MethodsUsing conditional gene targeting approach we have generated a transgenic 100% penetrant mouse model of CTCL wherein expression of a hyper-active STAT3 mutant protein (STAT3C) selectively in T lymphocytes results in skin pathology highly reminiscent of this human malignancy. To have a better understanding of the contribution of STAT3 in CD4 T cell differentiation program and CTCL, we used lymphocytes and splenocytes from 8 weeks old STAT3Cstopfl/fl (without CD4Cre, thus no pathology) and control YFPstopfl/fl (Yellow Fluorescent Protein floxed) mice (Figure 1a). We isolated naïve CD4 cells using Dynabeads and treated these CD4 cells with a transducible Cre enzyme (Tat-Cre) to delete the upstream stop cassette ex-vivo. These cells were then plated with stimulating α CD3/CD28 antibodies in various cytokine cocktails as shown in Figure 1b. These cells were harvested after 84 hours and stimulated with PMA/Inomycin for 4 hours for flowcytometric analysis. We are now using this in-vitro approach as well as our mouse model and primary tissue samples from CTCL patients to explore the impact of HDAC inhibitors on Th17/Treg balance. ResultsUpon Tat-Cre treatment we were able to analyze GFP+ (and therefore STAT3C expressing) vs. GFP- (control) T cells in the same differentiation conditions. Hyperactive STAT3C mutant protein favored TH17 differentiation as GFP Positive cells had significantly augmented IL17A production (Figure 1b). On the other hand, STAT3C expressing GFP positive cells in Th0 and Th1 differentiation conditions had less IFN-γ production compared to the GFP negative cells and control YFP fractions. Even the GFP negative fraction of STAT3C had less IFN-γ and more IL17A production than the control YFP fractions due to the cytokine milieu created by STAT3C expressing cells present in the same well. Conclusions:These results highlight an intriguing possibility that STAT3 dependent Th17 cells play a role in CTCL pathogenesis. Using our newly developed mouse model and CTCL patients' blood and skin samples (IRB approved), we are trying to distinguish whether Th17 cells are instigators of chronic inflammation that contributes to the malignant transformation of T cells or that Th17 cells may actually be the cells of origin in this malignancy. Our ongoing experiments also include the effects of HDACi on this ex-vivo CD4 T cell differentiation. In addition, we are studying the hallmarks of Th17 differentiation namely IL-17A, IL-22 and pSTAT3 positivity in the skin biopsy and blood samples of CTCL patients before and after treatment with HDACi. These experiments will help us determine the impact of HDAC inhibition on STAT3 activity and Th17 differentiation and have a better understanding of their mechanism of action in CTCL. Our hope is that our study will guide us to comprehend the contribution of chronic inflammation in carcinogenesis and potentially identify novel treatment targets and strategies in the field of hematological malignancies. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Cell cycle gene-specific control of transcription has a critical role in proliferation of primordial germ cells

Transcription elongation is stimulated by positive transcription elongation factor b (P-TEFb), for which activity is repressed in the 7SK small nuclear ribonucleoprotein (7SK snRNP) complex. We show here a critical role of 7SK snRNP in growth control of primordial germ cells (PGCs). The expression of p15(INK4b), a cyclin-dependent kinase inhibitor (CDKI) gene, in PGCs is selectively activated by P-TEFb and its recruiting molecule, Brd4, when the amount of active P-TEFb is increased due to reduction of the 7SK snRNP, and PGCs consequently undergo growth arrest. These results indicate that CDKI gene-specific control of transcription by 7SK snRNP plays a pivotal role in the maintenance of PGC proliferation.

Abstract PL03-03: The LKB1-AMPK pathway coordinates metabolic reprogramming with growth control

Abstract The serine/threonine kinase LKB1 is a tumor suppressor gene mutated in the familial cancer condition Peutz-Jeghers syndrome (PJS), as well as in 30% of sporadic non-small cell lung cancer (NSCLC). One of the critical substrates of LKB1 is the AMP-activated protein kinase (AMPK). AMPK is a highly conserved sensor of cellular energy status found in all eukaryotic cells that restores metabolic homeostasis following stress. Thus LKB1 is a unique energy-state sensitive regulator of growth and metabolic reprogramming via its effects on AMPK. Our laboratory has performed a three-pronged screen to identify novel substrates of AMPK that may mediate its effects on metabolism and growth control. These studies have led to the identification of components of the mTOR signaling pathway (raptor, TSC2), the autophagy pathway (ULK1), and transcriptional regulators of metabolism (Srebp1, HDAC4/5/7) all as direct substrates of AMPK. Collectively, these studies uncovered novel conserved effectors of LKB1 and AMPK that mediate their role as a metabolic checkpoint coordinating cell growth with energy status. The connection of AMPK to tumor suppression has also led many to examine whether compounds that normally serve to activate AMPK, may in fact exhibit anti-cancer activities. Notably, the most widely used type 2 diabetes therapeutic in the US and worldwide, metformin, is a mitochondrial OXPHOS inhibitor which activates AMPK. We have therefore examined the potential anti-cancer effects of metformin and its more potent analog phenformin, in the context of genetically engineered mouse models of lung cancer. Whereas cells containing an intact LKB1-AMPK pathway respond to this metabolic stress by undergoing growth arrest, cells lacking LKB1 are unable to sense the damage and continue to divide ultimately undergoing apoptosis. Thus we examined whether this class of compounds may show selective therapeutic efficacy in preclinical trials in Kras-dependent lung cancer mouse models with loss of LKB1 as compared to those bearing loss of p53. Citation Format: Reuben J. Shaw. The LKB1-AMPK pathway coordinates metabolic reprogramming with growth control. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr PL03-03.

N-(phosphonacetyl)-L-aspartate induces TAp73-dependent apoptosis by modulating multiple Bcl-2 proteins: potential for cancer therapy

p53 is essential for the cellular responses to DNA damage that help to maintain genomic stability. However, the great majority of human cancers undergo disruption of the p53-network. Identification and characterization of molecular components important in both p53-dependent and -independent apoptosis might be useful in developing novel therapies for cancers. In the complete absence of p53, cells treated with N-(phosphonacetyl)-L-aspartate (PALA) continue to synthesize DNA slowly and eventually progress through S phase, suffering severe DNA damage that in turn triggers apoptosis, whereas cells with functional p53 undergo growth arrest. In the present study, we investigated apoptotic signaling in response to PALA and the role of p53 expression in this pathway. We found that treatment of cells lacking p53 with PALA induced TAp73, Noxa, and Bim and inactivation of these proteins with dominant negative plasmids or siRNAs significantly inhibited apoptosis, suggesting that PALA-induced apoptosis was mediated via TAp73-dependent expression of Noxa and Bim. However, PALA treatment inhibited the expression of ΔNp73 only in cells lacking p53 but not in cells expressing p53. In addition, PALA treatment inhibited Bcl-2, and overexpression of Bcl-2 significantly inhibited PALA-induced apoptosis. Moreover, expression of p53 in these cells protected them from PALA-induced apoptosis by activating p21, sustaining the expression of ΔNp73 and inhibiting the induction of Noxa and Bim. Taken together, our study identifies novel but opposing roles for the p53 and TAp73 in the induction of Noxa and Bim and regulation of apoptosis. Our data will help to develop strategies to eliminate cancer cells lacking p53 while protecting normal cells with wild-type p53.

Combination of Valproic Acid and ATRA Restores RARβ2 Expression and Induces Differentiation in Cervical Cancer through the PI3K/Akt Pathway

Epigenetic silencing of the tumor suppressor gene, RARβ2, through histone deacetylation has been established as an important process of cervical carcinogenesis. This pivotal role has led to the suggestion that a combination of retinoids selective for RARβ2 with histone deacetylase (HDAC) inhibitors may have therapeutic potential. Valproic acid (VPA), a HDAC inhibitor, has a critical role in the regulation of gene expression through histone acetylation and causes transformed cells to undergo growth arrest, differentiation, and apoptosis. Therefore, we hypothesized that the combination of VPA and ATRA could restore RARβ2 expression, thus resulting in enhanced anti-neoplastic activity in cervical cancer. Here, we show that VPA combined with ATRA led to hyperacetylation of histone H3 and a significant alteration of gene expression in cervical cancer cells, including RARβ2 gene expression, which was upregulated 50- to 90-fold. The combination therapy effectively inhibited the growth of cervical cancer cells more than the single agent treatment both in vitro and in vivo. The additive effects were associated with a significant upregulation of p21(CIP1) and p53 as well as a pronounced decrease in p-Stat3. Furthermore, the combined treatment led to cell cycle arrest predominantly at the G1 phase, and it preferentially induced cell differentiation rather than apoptosis in cervical cancer cells. The differentiation program was determined by the presence of E-cadherinmediated adhesion and activation of the PI3K/Akt pathway. Taken together, these results provide new insight into the mechanisms of enhanced antitumor activity of the HDAC inhibitor and ATRA regimen, thus offering a new therapeutic strategy for cervical cancer patients.

Glycodelin-A interferes with IL-2/IL-2R signalling to induce cell growth arrest, loss of effector functions and apoptosis in T-lymphocytes

The progesterone-regulated glycoprotein glycodelin-A (GdA), secreted by the decidualized endometrium at high concentrations in primates, inhibits the maternal immune response against fetal antigens and thereby contributes to the tolerance of the semi-allogenic fetus during a normal pregnancy. Our earlier studies demonstrated the ability of GdA to induce an intrinsic apoptotic cascade in CD4(+) T-lymphocytes and suppress the cytolytic effector function of CD8(+) T-lymphocytes. In this report, we investigated further into the mechanism of action of GdA controlling perforin and granzyme B expression in CD8(+) T-lymphocytes and the mechanism of action of GdA leading to lymphocyte death. Flow cytometry analysis was performed to check for the surface expression of interleukin-2 receptor α (IL-2Rα) and intracellular eomesodermin (Eomes) in activated T-lymphocytes, whereas quantitative RT-PCR analysis was used to find out their mRNA profile upon GdA treatment. Western analysis was carried out to confirm the protein level of Bax and Bcl-2. GdA reduces the surface expression of the high-affinity IL-2R complex by down-regulating the synthesis of IL-2Rα (CD25). This disturbs the optimal IL-2 signalling and decreases the Eomes expression, which along with IL-2 directly regulates perforin and granzymes expression. Consequently, the CD8(+) T-lymphocytes undergo growth arrest and are unable to mature into competent cytotoxic T-lymphocytes. In the CD4(+) T-lymphocytes, growth factor IL-2 deprivation leads to proliferation inhibition, decreased Bcl-2/enhanced Bax expression, culminating in mitochondrial stress and cell death. GdA spurs cell cycle arrest, loss of effector functions and apoptosis in different T-cell subsets by making T-lymphocytes unable to respond to IL-2.

Modelling genetic and clinical heterogeneity in epithelial ovarian cancers

The biology underlying early-stage epithelial ovarian cancer (EOC) development is poorly understood. Identifying biomarkers associated with early-stage disease could have a significant impact on reducing mortality. Here, we describe establishment of a three-dimensional (3D) in vitro genetic model of EOC initiation and early-stage neoplastic progression. Normal primary ovarian epithelial (POE) cells, immortalized using hTERT (immortalised ovarian epithelial [IOE] cells), were partially transformed by overexpressing the CMYC oncogene (IOE(CMYC) cells). Subsequent expression of mutant alleles of KRAS (KRAS(G12V)) or BRAF (BRAF(V600E)) created double-mutant lines (IOE(CMYC.KRAS) and IOE(CMYC.BRAF)). The transformed phenotype of IOE(CMYC) cells was further enhanced in concert with KRAS(G12V)/BRAF(V600E) expression, as in vitro analyses indicated that IOE(CMYC) cells had undergone morphological and phenotypic changes characteristic of neoplastic progression. When cultured as 3D spheroids, IOE cells underwent growth arrest, reminiscent of nonproliferative, unstimulated POE in vivo. In contrast, IOSE(CMYC+BRAF/KRAS) cells formed highly proliferative, poly-aggregate spheroid structures, showing increased expression of the Wilms tumour 1 tumourigenic marker and MIB1 proliferation marker. Transcriptomic analyses identified different gene expression profiles between the different cell lines and novel candidate genes (e.g. RGS4, CTGF and THBS1) that are somatically altered in EOCs. Gene expression signatures were compared with signatures from primary EOCs; tumours with IOE(CMYC) 'like' signatures were more likely to be high grade (P = 0.018); tumours with BRAF signatures were associated with improved relapse-free survival (P = 0.003). In conclusion, we have established in vitro 3D models of early-stage EOCs, which reflect genetic and phenotypic heterogeneity of the disease. Molecular genetic characteristics of these models correlated with molecular and clinical features of primary EOCs.

Oncogenic Fusion Protein EWS/FLI1 Down-regulates Gene Expression by Both Transcriptional and Posttranscriptional Mechanisms

Ewing family tumors are characterized by a translocation between the RNA binding protein EWS and one of five ETS transcription factors, most commonly FLI1. The fusion protein produced by the translocation has been thought to act as an aberrant transcription factor, leading to changes in gene expression and cellular transformation. In this study, we investigated the specific processes EWS/FLI1 utilizes to alter gene expression. Using both heterologous NIH 3T3 and human Ewing Family Tumor cell lines, we have demonstrated by quantitative pre-mRNA analysis that EWS/FLI1 repressed the expression of previously validated direct target genes at the level of transcript synthesis. ChIP experiments showed that EWS/FLI1 decreases the amount of Pol II at the promoter of down-regulated genes in both murine and human model systems. However, in down-regulated target genes, there was a significant disparity between the modulation of cognate mRNA and pre-mRNAs, suggesting that these genes could also be regulated at a posttranscriptional level. Confirming this, we found that EWS/FLI1 decreased the transcript half-life of insulin-like growth factor binding protein 3, a down-regulated direct target gene in human tumor-derived Ewing's sarcoma cell lines. Additionally, we have shown through reexpression experiments that full EWS/FLI1-mediated transcriptional repression requires intact EWS and ETS domains. Together these data demonstrate that EWS/FLI1 can dictate steady-state target gene expression by modulating both transcript synthesis and degradation.

Nevus senescence.

Melanomas and nevi share many of the same growth-promoting mutations. However, melanomas grow relentlessly while benign nevi eventually undergo growth arrest and stabilize. The difference in their long-term growth potential may be attributed to activation of cellular senescence pathways. The primary mediator of senescence in nevi appears to be p16. Redundant, secondary senescence systems are also present and include the p14-p53-p21 pathway, the IGFBP7 pathway, the FBXO31 pathway, and the PI3K mediated stress induced endoplasmic reticulum unfolded protein response. It is evident that these senescence pathways result in an irreversible arrest in most instances; however, they can clearly be overcome in melanoma. Circumvention of these pathways is most frequently associated with gene deletion or transcriptional repression. Reactivation of senescence mechanisms could serve to inhibit melanoma tumor progression.

Acquisition of p53 mutations in response to the non-genotoxic p53 activator Nutlin-3

Wild-type p53 is a stress-responsive tumor suppressor and potent growth inhibitor. Genotoxic stresses (e.g. ionizing and UV radiation or chemotherapeutic drug treatment) can activate p53, but also induce mutations in the P53 gene and thus select for p53-mutated cells. Nutlin-3a (Nutlin) is pre-clinical drug that activates p53 in a non-genotoxic fashion. Nutlin occupies the p53-binding pocket of MDM2, activating p53 by blocking the p53-MDM2 interaction. Because Nutlin neither binds p53 directly nor introduces DNA damage, we hypothesized Nutlin would not induce P53 mutations and therefore not select for p53-mutated cells. To test this, populations of SJSA-1 (p53 wild-type) cancer cells were expanded that survived repeated Nutlin exposures, and individual clones were isolated. Group 1 clones were resistant to Nutlin-induced apoptosis, but still underwent growth-arrest. Surprisingly, while some Group 1 clones retained wild-type p53, others acquired a heterozygous p53 mutation. Apoptosis resistance in Group 1 clones was associated with decreased PUMA induction and decreased caspase 3/7 activation. Group 2 clones were resistant to both apoptosis and growth-arrest induced by Nutlin. Group 2 clones had acquired mutations in the p53 DNA-binding domain and expressed only mutant p53s that were induced by Nutlin treatment, but were unable to bind the P21 and PUMA gene promoters, and unable to activate transcription. These results demonstrate that non-genotoxic p53 activation (e.g. by Nutlin treatment) can lead to the acquisition of somatic mutations in p53 and select for p53-mutated cells. These findings have implications for the potential clinical use of Nutlin and other small molecule MDM2 antagonists.

Interplay between FGF2 and BMP controls the self-renewal, dormancy and differentiation of rat neural stem cells

Mouse and human central nervous system progenitor cells can be propagated extensively ex vivo as stem cell lines. For the rat, however, in vitro expansion has proven to be problematic owing to proliferation arrest and differentiation. Here, we analyse the establishment, in adherent culture, of undifferentiated tripotent neural stem (NS) cell lines derived from rat foetal brain and spinal cord. Rat NS cells invariably undergo growth arrest and apparent differentiation after several passages; however, conditioned medium from proliferating cultures can overcome this block, enabling continuous propagation of undifferentiated rat NS cells. We found that dormancy is induced by autocrine production of bone morphogenetic proteins (BMPs). Accordingly, the BMP antagonist noggin can replace conditioned medium to sustain continuous self-renewal. Noggin can also induce dormant cells to re-enter the cell cycle, upon which they reacquire neurogenic potential. We further show that fibroblast growth factor 2 (FGF2) is required to suppress terminal astrocytic differentiation and maintain stem cell potency during dormancy. These findings highlight an extrinsic regulatory network, comprising BMPs, BMP antagonists and FGF2 signals, that governs the proliferation, dormancy and differentiation of rat NS cells and which can be manipulated to enable long-term clonogenic self-renewal.

Interplay between FGF2 and BMP controls the self-renewal, dormancy and differentiation of rat neural stem cells.

Mouse and human central nervous system progenitor cells can be propagated extensively ex vivo as stem cell lines. For the rat, however, in vitro expansion has proven to be problematic owing to proliferation arrest and differentiation. Here, we analyse the establishment, in adherent culture, of undifferentiated tripotent neural stem (NS) cell lines derived from rat foetal brain and spinal cord. Rat NS cells invariably undergo growth arrest and apparent differentiation after several passages; however, conditioned medium from proliferating cultures can overcome this block, enabling continuous propagation of undifferentiated rat NS cells. We found that dormancy is induced by autocrine production of bone morphogenetic proteins (BMPs). Accordingly, the BMP antagonist noggin can replace conditioned medium to sustain continuous self-renewal. Noggin can also induce dormant cells to re-enter the cell cycle, upon which they reacquire neurogenic potential. We further show that fibroblast growth factor 2 (FGF2) is required to suppress terminal astrocytic differentiation and maintain stem cell potency during dormancy. These findings highlight an extrinsic regulatory network, comprising BMPs, BMP antagonists and FGF2 signals, that governs the proliferation, dormancy and differentiation of rat NS cells and which can be manipulated to enable long-term clonogenic self-renewal.

Loss of Cystic Fibrosis Transmembrane Conductance Regulator Function Enhances Activation of p38 and ERK MAPKs, Increasing Interleukin-6 Synthesis in Airway Epithelial Cells Exposed to Pseudomonas aeruginosa

In cystic fibrosis (CF), the absence of functional cystic fibrosis transmembrane conductance regulator (CFTR) translates into chronic bacterial infection, excessive inflammation, tissue damage, impaired lung function and eventual death. Understanding the mechanisms underlying this vicious circle of inflammation is important to design better therapies for CF. We found in CF lung biopsies increased immunoreactivity for p38 MAPK activity markers. Moreover, when compared with their non-CF counterpart, airway epithelial cells expressing the most common mutation in CF (CFTRDeltaF508) were more potent at inducing neutrophil chemotaxis through increased interleukin (IL)-6 synthesis when challenged with Pseudomonas aeruginosa diffusible material. We then discovered that in CFTRDeltaF508 cells, the p38 and ERK MAPKs are hyperactivated in response to P. aeruginosa diffusible material, leading to increased IL-6 mRNA expression and stability. Moreover, although TLR5 contributes to p38 MAPK activation upon P. aeruginosa challenge, it only played a weak role in IL-6 synthesis. Instead, we found that the production of reactive oxygen species is essential for IL-6 synthesis in response to P. aeruginosa diffusible material. Finally, we uncovered that in CFTRDeltaF508 cells, the extracellular glutathione levels are decreased, leading to a greater sensitivity to reactive oxygen species, providing an explanation for the hyperactivation of the p38 and ERK MAPKs and increased IL-6 synthesis. Taken together, our study has characterized a mechanism whereby the CFTRDeltaF508 mutation in airway epithelial cells contributes to increase inflammation of the airways.