Selective repression of retinoic acid target genes by RIP140 during induced tumor cell differentiation of pluripotent human embryonal carcinoma cells

<p>Abstract</p> <p>Background</p> <p>The use of retinoids as anti-cancer agents has been limited due to resistance and low efficacy. The dynamics of nuclear receptor coregulation are incompletely understood. Cell-and context-specific activities of nuclear receptors may be in part due to distinct coregulator complexes recruited to distinct subsets of target genes. RIP140 (also called NRIP1) is a ligand-dependent corepressor that is inducible with retinoic acid (RA). We had previously shown that RIP140 limits RA induced tumor cell differentiation of embryonal carcinoma; the pluriopotent stem cells of testicular germ cell tumors. This implies that RIP140 represses key genes required for RA-mediated tumor cell differentiation. Identification of these genes would be of considerable interest.</p> <p>Results</p> <p>To begin to address this issue, microarray technology was employed to elucidate in a <it>de novo </it>fashion the global role of RIP140 in RA target gene regulation of embryonal carcinoma. Subclasses of genes were affected by RIP140 in distinct manners.</p> <p>Interestingly, approximately half of the RA-dependent genes were unaffected by RIP140. Hence, RIP140 appears to discriminate between different classes of RA target genes. In general, RIP140-dependent gene expression was consistent with RIP140 functioning to limit RA signaling and tumor cell differentiation. Few if any genes were regulated in a manner to support a role for RIP140 in "active repression". We also demonstrated that RIP140 silencing sensitizes embryonal carcinoma cells to low doses of RA.</p> <p>Conclusion</p> <p>Together the data demonstrates that RIP140 has profound effects on RA-mediated gene expression in this cancer stem cell model. The RIP140-dependent RA target genes identified here may be particularly important in mediating RA-induced tumor cell differentiation and the findings suggest that RIP140 may be an attractive target to sensitize tumor cells to retinoid-based differentiation therapy. We discuss these data in the context of proposed models of RIP140-mediated repression.</p

Evidence for Tankyrases as Antineoplastic Targets in Lung Cancer

Background: New pharmacologic targets are urgently needed to treat or prevent lung cancer, the most common cause of cancer death for men and women. This study identified one such target. This is the canonical Wnt signaling pathway, which is deregulated in cancers, including those lacking adenomatous polyposis coli or β -catenin mutations. Two poly-ADP-ribose polymerase (PARP) enzymes regulate canonical Wnt activity: tankyrase (TNKS) 1 and TNKS2. These enzymes poly-ADP-ribosylate (PARsylate) and destabilize axin, a key component of the β -catenin phosphorylation complex. Methods: This study used comprehensive gene profiles to uncover deregulation of the Wnt pathway in murine transgenic and human lung cancers, relative to normal lung. Antineoplastic consequences of genetic and pharmacologic targeting of TNKS in murine and human lung cancer cell lines were explored, and validated in vivo in mice by implantation of murine transgenic lung cancer cells engineered with reduced TNKS expression relative to controls. Results: Microarray analyses comparing Wnt pathway members in malignant versus normal tissues of a murine transgenic cyclin E lung cancer model revealed deregulation of Wnt pathway components, including TNKS1 and TNKS2. Real-time PCR assays independently confirmed these results in paired normal-malignant murine and human lung tissues. Individual treatments of a panel of human and murine lung cancer cell lines with the TNKS inhibitors XAV939 and IWR-1 dose-dependently repressed cell growth and increased cellular axin 1 and tankyrase levels. These inhibitors also repressed expression of a Wnt-responsive luciferase construct, implicating the Wnt pathway in conferring these antineoplastic effects. Individual or combined knockdown of TNKS1 and TNKS2 with siRNAs or shRNAs reduced lung cancer cell growth, stabilized axin, and repressed tumor formation in murine xenograft and syngeneic lung cancer models. Conclusions: Findings reported here uncovered deregulation of specific components of the Wnt pathway in both human and murine lung cancer models. Repressing TNKS activity through either genetic or pharmacological approaches antagonized canonical Wnt signaling, reduced murine and human lung cancer cell line growth, and decreased tumor formation in mouse models. Taken together, these findings implicate the use of TNKS inhibitors to target the Wnt pathway to combat lung cancer

Repression of cyclin D1 as a target for germ cell tumors

Metastatic germ cell tumors (GCT) are curable, however GCTs refractory to cisplatin-based chemotherapy have a poor prognosis. This study explores D-type cyclins as molecular targets in GCTs because all-trans-retinoic acid (RA)-mediated differentiation of the human embryonal carcinoma (EC) cell line NT2/D1 is associated with G1 cell cycle arrest and proteasomal degradation of cyclin D1. RA effects on D-type cyclins are compared in human EC cells that are RA sensitive or dually RA and cisplatin resistant (NT2/D1-R1) and in clinical GCTs that have both EC and mature teratoma components. Notably, GCT differentiation was associated with reduced cyclin D1 but increased cyclin D3 expression. RA was shown here to repress cyclin D1 through a transcriptional mechanism in addition to causing its degradation. The siRNA-mediated repression of individual cyclin D species resulted in growth inhibition in both RA sensitive and resistant EC cells. Only repression of cyclin D1 occurred in vitro and when clinical GCTs mature, implicating cyclin D1 as a molecular therapeutic target. To confirm this, the EGFR-tyrosine kinase inhibitor, Erlotinib, was used to repress cyclin D1. This inhibited proliferation in RA and cisplatin sensitive and resistant EC cells. Taken together, these findings implicate cyclin D1 targeting agents for the treatment of GCTs

Refractory testicular germ cell tumors are highly sensitive to the second generation DNA methylation inhibitor guadecitabine

Testicular germ cell tumors (TGCTs) are the most common cancers of young males. A substantial portion of TGCT patients are refractory to cisplatin. There are no effective therapies for these patients, many of whom die from progressive disease. Embryonal carcinoma (EC) are the stem cells of TGCTs. In prior in vitro studies we found that EC cells were highly sensitive to the DNA methyltransferase inhibitor, 5-aza deoxycytidine (5-aza). Here, as an initial step in bringing demethylation therapy to the clinic for TGCT patients, we evaluated the effects of the clinically optimized, second generation demethylating agent guadecitabine (SGI-110) on EC cells in an animal model of cisplatin refractory testicular cancer. EC cells were exquisitely sensitive to guadecitabine and the hypersensitivity was dependent on high levels of DNA methyltransferase 3B. Guadecitabine mediated transcriptional reprogramming of EC cells included induction of p53 targets and repression of pluripotency genes. As a single agent, guadecitabine completely abolished progression and induced complete regression of cisplatin resistant EC xenografts even at doses well below those required to impact somatic solid tumors. Low dose guadecitabine also sensitized refractory EC cells to cisplatin in vivo. Genome-wide analysis indicated that in vivo antitumor activity was associated with activation of p53 and immune-related pathways and the antitumor effects of guadecitabine were dependent on p53, a gene rarely mutated in TGCTs. These preclinical findings suggest that guadecitabine alone or in combination with cisplatin is a promising strategy to treat refractory TGCT patients

GHTD-amide : A naturally occurring beta cell-derived peptide with hypoglycemic activity

in the early 1970s, a peptide fraction with insulin potentiating activity was purified from human urine but the identity and origins of the active constituent remained unknown. Here we identify the active component and characterize its origins. The active peptide was identified as an alpha amidated tetrapeptide with the sequence GHTD-amide. The peptide was synthesized and tested for stimulation of glycogen synthesis and insulin potentiation by insulin tolerance testing in insulin-deficient rats, which confirmed GHTD-amide as the active peptide. Tissue localization using a peptide-specific anti-serum and epifluorescent and confocal microscopy showed decoration of pancreatic islets but not other tissues. Confocal microscopy revealed co-localization with insulin and immunogold and electron microscopy showed localization to dense core secretory granules. Consistent with these observations GHTD-amide was found in media conditioned by MIN6 islet beta cells. Sequence database searching found no annotated protein in the human proteome encoding a potential precursor for GHTD-amide. We conclude that the insulin potentiating activity originally described in human urine is attributable to the tetrapeptide GHTD-amide. GHTD-amide is a novel peptide produced by pancreatic beta cells and no precursor protein is present in the annotated human proteome. Stimulation of glycogen synthesis and co-localization with insulin in beta cells suggest that GHTD-amide may play a role in glucose homeostasis by enhancing insulin action and glucose storage in tissues. (C) 2008 Elsevier Inc. All rights reserved

Hypermethylation and global remodelling of DNA methylation is associated with acquired cisplatin resistance in testicular germ cell tumours

Testicular germ cell tumours (TGCTs) respond well to cisplatin-based therapy. However, cisplatin resistance and poor outcomes do occur. It has been suggested that a shift towards DNA hypermethylation mediates cisplatin resistance in TGCT cells, although there is little direct evidence to support this claim. Here we utilized a series of isogenic cisplatin-resistant cell models and observed a strong association between cisplatin resistance in TGCT cells and a net increase in global CpG and non-CpG DNA methylation spanning regulatory, intergenic, genic and repeat elements. Hypermethylated loci were significantly enriched for repressive DNA segments, CTCF and RAD21 sites and lamina associated domains, suggesting that global nuclear reorganization of chromatin structure occurred in resistant cells. Hypomethylated CpG loci were significantly enriched for EZH2 and SUZ12 binding and H3K27me3 sites. Integrative transcriptome and methylome analyses showed a strong negative correlation between gene promoter and CpG island methylation and gene expression in resistant cells and a weaker positive correlation between gene body methylation and gene expression. A bidirectional shift between gene promoter and gene body DNA methylation occurred within multiple genes that was associated with upregulation of polycomb targets and downregulation of tumour suppressor genes. These data support the hypothesis that global remodelling of DNA methylation is a key factor in mediating cisplatin hypersensitivity and chemoresistance of TGCTs and furthers the rationale for hypomethylation therapy for refractory TGCT patients

Wnt pathway reprogramming during human embryonal carcinoma differentiation and potential for therapeutic targeting

<p>Abstract</p> <p>Background</p> <p>Testicular germ cell tumors (TGCTs) are classified as seminonas or non-seminomas of which a major subset is embryonal carcinoma (EC) that can differentiate into diverse tissues. The pluripotent nature of human ECs resembles that of embryonic stem (ES) cells. Many Wnt signalling species are regulated during differentiation of TGCT-derived EC cells. This study comprehensively investigated expression profiles of Wnt signalling components regulated during induced differentiation of EC cells and explored the role of key components in maintaining pluripotency.</p> <p>Methods</p> <p>Human embryonal carcinoma cells were stably infected with a lentiviral construct carrying a canonical Wnt responsive reporter to assess Wnt signalling activity following induced differentiation. Cells were differentiated with all-<it>trans </it>retinoic acid (RA) or by targeted repression of pluripotency factor, POU5F1. A Wnt pathway real-time-PCR array was used to evaluate changes in gene expression as cells differentiated. Highlighted Wnt pathway genes were then specifically repressed using siRNA or stable shRNA and transfected EC cells were assessed for proliferation, differentiation status and levels of core pluripotency genes.</p> <p>Results</p> <p>Canonical Wnt signalling activity was low basally in undifferentiated EC cells, but substantially increased with induced differentiation. Wnt pathway gene expression levels were compared during induced differentiation and many components were altered including ligands (WNT2B), receptors (FZD5, FZD6, FZD10), secreted inhibitors (SFRP4, SFRP1), and other effectors of Wnt signalling (FRAT2, DAAM1, PITX2, Porcupine). Independent repression of FZD5, FZD7 and WNT5A using transient as well as stable methods of RNA interference (RNAi) inhibited cell growth of pluripotent NT2/D1 human EC cells, but did not appreciably induce differentiation or repress key pluripotency genes. Silencing of FZD7 gave the greatest growth suppression in all human EC cell lines tested including NT2/D1, NT2/D1-R1, Tera-1 and 833K cells.</p> <p>Conclusion</p> <p>During induced differentiation of human EC cells, the Wnt signalling pathway is reprogrammed and canonical Wnt signalling induced. Specific species regulating non-canonical Wnt signalling conferred growth inhibition when targeted for repression in these EC cells. Notably, FZD7 repression significantly inhibited growth of human EC cells and is a promising therapeutic target for TGCTs.</p

Lung cancer diagnosed following an emergency admission: exploring patient and carer perspectives on delay in seeking help

Purpose Compared to others, patients diagnosed with lung cancer following an emergency, unplanned admission to hospital (DFEA) have more advanced disease and poorer prognosis. Little is known about DFEA patients’ beliefs about cancer and its symptoms or about their help-seeking behaviours prior to admission. Methods As part of a larger single-centre, prospective mixed-methods study conducted in one University hospital, we undertook qualitative interviews with patients DFEA and their carers to obtain their understanding of symptoms and experiences of trying to access healthcare services before admission to hospital. Interviews were recorded and transcribed. Framework analysis was employed. Results Thirteen patients and 10 carers plus 3 bereaved carers took part in interviews. Three patient/carer dyads were interviewed together. Participants spoke about their symptoms and why they did not seek help sooner. They described complex and nuanced experiences. Some (n = 12) had what they recalled as the wrong symptoms for lung cancer and attributed them either to a pre-existing condition or to ageing. In other cases (n = 9), patients or carers realised with hindsight that their symptoms were signs of lung cancer, but at the time had made other attributions to account for them. In some cases (n = 3), a sudden onset of symptoms was reported. Some GPs (n = 6) were also reported to have made incorrect attributions about cause. Conclusion Late diagnosis meant that patients DFEA needed palliative support sooner after diagnosis than patients not DFEA. Professionals and lay people interpret health and illness experiences differently

Retinoic Acid Mediates Long-Paced Oscillations in Retinoid Receptor Activity: Evidence for a Potential Role for RIP140

Mechanisms that underlie oscillatory transcriptional activity of nuclear receptors (NRs) are incompletely understood. Evidence exists for rapid, cyclic recruitment of coregulatory complexes upon activation of nuclear receptors. RIP140 is a NR coregulator that represses the transactivation of agonist-bound nuclear receptors. Previously, we showed that RIP140 is inducible by all-trans retinoic acid (RA) and mediates limiting, negative-feedback regulation of retinoid signaling.Here we report that in the continued presence of RA, long-paced oscillations of retinoic acid receptor (RAR) activity occur with a period ranging from 24 to 35 hours. Endogenous expression of RIP140 and other RA-target genes also oscillate in the presence of RA. Cyclic retinoid receptor transactivation is ablated by constitutive overexpression of RIP140. Further, depletion of RIP140 disrupts cyclic expression of the RA target gene HOXA5. Evidence is provided that RIP140 may limit RAR signaling in a selective, non-redundant manner in contrast to the classic NR coregulators NCoR1 and SRC1 that are not RA-inducible, do not cycle, and may be partially redundant in limiting RAR activity. Finally, evidence is provided that RIP140 can repress and be induced by other nuclear receptors in a manner that suggests potential participation in other NR oscillations.We provide evidence for novel, long-paced oscillatory retinoid receptor activity and hypothesize that this may be paced in part, by RIP140. Oscillatory NR activity may be involved in mediating hormone actions of physiological and pathological importance

Designing clinical trials for assessing the effects of cognitive training and physical activity interventions on cognitive outcomes: The Seniors Health and Activity Research Program Pilot (SHARP-P) Study, a randomized controlled trial

<p>Abstract</p> <p>Background</p> <p>The efficacy of non-pharmacological intervention approaches such as physical activity, strength, and cognitive training for improving brain health has not been established. Before definitive trials are mounted, important design questions on participation/adherence, training and interventions effects must be answered to more fully inform a full-scale trial.</p> <p>Methods</p> <p>SHARP-P was a single-blinded randomized controlled pilot trial of a 4-month physical activity training intervention (PA) and/or cognitive training intervention (CT) in a 2 × 2 factorial design with a health education control condition in 73 community-dwelling persons, aged 70-85 years, who were at risk for cognitive decline but did not have mild cognitive impairment.</p> <p>Results</p> <p>Intervention attendance rates were higher in the CT and PACT groups: CT: 96%, PA: 76%, PACT: 90% (p=0.004), the interventions produced marked changes in cognitive and physical performance measures (p≤0.05), and retention rates exceeded 90%. There were no statistically significant differences in 4-month changes in composite scores of cognitive, executive, and episodic memory function among arms. Four-month improvements in the composite measure increased with age among participants assigned to physical activity training but decreased with age for other participants (intervention*age interaction p = 0.01). Depending on the choice of outcome, two-armed full-scale trials may require fewer than 1,000 participants (continuous outcome) or 2,000 participants (categorical outcome).</p> <p>Conclusions</p> <p>Good levels of participation, adherence, and retention appear to be achievable for participants through age 85 years. Care should be taken to ensure that an attention control condition does not attenuate intervention effects. Depending on the choice of outcome measures, the necessary sample sizes to conduct four-year trials appear to be feasible.</p> <p>Trial Registration</p> <p>Clinicaltrials.gov Identifier: <a href="http://www.clinicaltrials.gov/ct2/show/NCT00688155">NCT00688155</a></p