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Prostate cancer (PCa) commonly metastasizes to the bone where the cells frequently undergo dormancy. The escape of disseminated tumor cells from cellular dormancy is a major cause of recurrence in marrow. Abscisic acid (ABA), a phytohormone, is known to regulate dormancy of plant seeds and to regulate other stress responses in plants. Recently, ABA was found to be synthesized by mammals cells and has been linked to human disease. Yet the role of ABA in regulating tumor dormancy or reactivation is unknown. We found that ABA is produced by human marrow cells, and exogenous ABA inhibits PCa cell proliferation while increasing the expression of p27, p21, and p16 and decreasing the expression of the proliferation marker, Ki67. Further, ABA significantly increased the percentage of PCa cells in the G0 phase of the cell cycle as well as the duration the cells were arrested in G0. We found that ABA regulates an increase of PPARγ receptor expression and suppressed phosphorylation of mTOR/p70S6K signaling and resulting in the induction of the cellular dormancy. We then confirmed that ABA regulates G0 cell cycle arrest through PPARγ receptor signaling in vitro and under co-culture conditions with osteoblasts. Finally, we demonstrate that ABA regulates PCa dormancy in vivo following intratibial injection in an animal model. Together these data suggest that the ABA and PPARγ signaling pathways contribute to the establishment of PCa cellular dormancy in the bone marrow microenvironment. These findings may suggest critical pathways for targeting metastatic disease.

Background Disseminated tumor cells (DTCs) have been reported in the bone marrow (BM) of patients with localized prostate cancer (PCa). However, the existence of these cells continues to be questioned, and few methods exist for viable DTC isolation. Therefore, we sought to develop novel approaches to identify and, if detected, analyze localized PCa patient DTCs. Methods We used fluorescence-activated cell sorting (FACS) to isolate a putative DTC population, which was negative for CD45, CD235a, alkaline phosphatase, and CD34, and strongly expressed EPCAM. We examined tumor cell content by bulk cell RNA sequencing (RNA-Seq) and whole-exome sequencing after whole genome amplification. We also enriched for BM DTCs with α-EPCAM immunomagnetic beads and performed quantitative reverse trancriptase polymerase chain reaction (qRT-PCR) for PCa markers. Results At a threshold of 4 cells per million BM cells, the putative DTC population was present in 10 of 58 patients (17%) with localized PCa, 4 of 8 patients with metastatic PCa of varying disease control, and 1 of 8 patients with no known cancer, and was positively correlated with patients' plasma PSA values. RNA-Seq analysis of the putative DTC population collected from samples above (3 patients) and below (5 patients) the threshold of 4 putative DTCs per million showed increased expression of PCa marker genes in 4 of 8 patients with localized PCa, but not the one normal donor who had the putative DTC population present. Whole-exome sequencing also showed the presence of single nucleotide polymorphisms and structural variants in the gene characteristics of PCa in 2 of 3 localized PCa patients. To examine the likely contaminating cell types, we used a myeloid colony formation assay, differential counts of cell smears, and analysis of the RNA-Seq data using the CIBERSORT algorithm, which most strongly suggested the presence of B-cell lineages as a contaminant. Finally, we used EPCAM enrichment and qRT-PCR for PCa markers to estimate DTC prevalence and found evidence of DTCs in 21 of 44 samples (47%). Conclusion These data support the presence of DTCs in the BM of a subset of patients with localized PCa and describe a novel FACS method for isolation and analysis of viable DTCs.

Scrapie resistance or susceptibility in sheep is associated with single nucleotide polymorphisms (SNPs) at codons 136, 154, and 171 of the prion protein gene ( PRNP). In addition, phenylalanine mutation at codon 141 has been recognized as a risk factor for atypical scrapie. In contrast, K222, D146, and S146 alleles confer genetic resistance to classical scrapie in goats. High-throughput genotyping technologies would provide significant benefits in scrapie eradication plans. The ability to resolve oligonucleotides varying in mass by less than a single nucleotide makes MALDI-TOF mass spectrometry (MS) a suitable platform for PRNP genotyping. We evaluated the commercial Myriapod scrapie kit (Diatech Pharmacogenetics), associated with a highly automated processing platform incorporating MALDI-TOF MS technology, to detect SNPs at codons 136, 154, 171, 141, and 222 of small ruminant PRNP. The Myriapod scrapie kit was accredited according to UNI CEI EN ISO/IEC 17025. We present the genotyping results of 10,960 sheep in Sicily and 1,822 goats in Sicily and Calabria (southern Italy) tested during 2017. We found a high frequency (43.9%) of the protective ARR allele in sheep and a promising 12.3% of the resistant K222 variant in goats. This efficient and high-throughput method is suitable for extensive PRNP genotyping, as requested in the European scrapie eradication plan.

Simple Summary Urothelial bladder cancer (BC) is one of the most fatal cancers, with a dismal five-year survival rate of 5% in patients with metastatic disease. Clinically relevant benefits of immunotherapy in advanced or metastatic bladder cancer have led to Food and Drug Administration (FDA) approval of immune checkpoint inhibitors (ICIs) as second- or first-line therapy in patients unresponsive to or ineligible for standard treatment. The advantage of ICIs is being investigated in various stages of BC, either as monotherapy or in combination with other drugs. In this review we discuss the role of ICIs in BC, highlighting their current clinical application and outlining future therapeutic perspectives. Abstract Bladder cancer (BC) is the most common malignancy of the genitourinary tract, with high morbidity and mortality rates. Until recently, the treatment of locally advanced or metastatic urothelial BC was based on the use of chemotherapy alone. Since 2016, five immune checkpoint inhibitors (ICIs) have been approved by the Food and Drug Administration (FDA) in different settings, i.e., first-line, maintenance and second-line treatment, while several trials are still ongoing in the perioperative context. Lately, pembrolizumab, a programmed death-1 (PD-1) inhibitor, has been approved for Bacillus Calmette–Guérin (BCG)-unresponsive high-risk non-muscle invasive bladder cancer (NMIBC), using immunotherapy at an early stage of the disease. This review investigates the current state and future perspectives of immunotherapy in BC, focusing on the rationale and results of combining immunotherapy with other therapeutic strategies.

Terminally differentiated cells of the nervous system have long been considered to be in a stable non-cycling state and are often considered to be permanently in G0. Exit from the cell cycle during development is often coincident with the differentiation of neurons, and is critical for neuronal function. But what happens in long lived postmitotic tissues that accumulate cell damage or suffer cell loss during aging? In other contexts, cells that are normally non-dividing or postmitotic can or re-enter the cell cycle and begin replicating their DNA to facilitate cellular growth in response to cell loss. This leads to a state called polyploidy, where cells contain multiple copies of the genome. A growing body of literature from several vertebrate and invertebrate model organisms has shown that polyploidy in the nervous system may be more common than previously appreciated and occurs under normal physiological conditions. Moreover, it has been found that neuronal polyploidization can play a protective role when cells are challenged with DNA damage or oxidative stress. By contrast, work over the last two and a half decades has discovered a link between cell-cycle reentry in neurons and several neurodegenerative conditions. In this context, neuronal cell cycle re-entry is widely considered to be aberrant and deleterious to neuronal health. In this review, we highlight historical and emerging reports of polyploidy in the nervous systems of various vertebrate and invertebrate organisms. We discuss the potential functions of polyploidization in the nervous system, particularly in the context of long-lived cells and age-associated polyploidization. Finally, we attempt to reconcile the seemingly disparate associations of neuronal polyploidy with both neurodegeneration and neuroprotection.

Using a gain-of-function screen in Drosophila, we identified the Kruppel-like factor Cabut (Cbt) as a positive regulator of cell cycle gene expression and cell proliferation. Enforced cbt expression is sufficient to induce an extra cell division in the differentiating fly wing or eye, and also promotes intestinal stem cell divisions in the adult gut. Although inappropriate cell proliferation also results from forced expression of the E2f1 transcription factor or its target, Cyclin E, Cbt does not increase E2F1 or Cyclin E activity. Instead, Cbt regulates a large set of E2F1 target genes independently of E2F1, and our data suggest that Cbt acts via distinct binding sites in target gene promoters. Although Cbt was not required for cell proliferation during wing or eye development, Cbt is required for normal intestinal stem cell divisions in the midgut, which expresses E2F1 at relatively low levels. The E2F1-like functions of Cbt identify a distinct mechanism for cell cycle regulation that may be important in certain normal cell cycles, or in cells that cycle inappropriately, such as cancer cells.

Long-lived cells such as terminally differentiated postmitotic neurons and glia must cope with the accumulation of damage over the course of an animal’s lifespan. How long-lived cells deal with ageing-related damage is poorly understood. Here we show that polyploid cells accumulate in the ageing adult fly brain and that polyploidy protects against DNA damage-induced cell death. Multiple types of neurons and glia that are diploid at eclosion, become polyploid with age in the adultDrosophilabrain. The optic lobes exhibit the highest levels of polyploidy, associated with an elevated DNA damage response in this brain region with age. Inducing oxidative stress or exogenous DNA damage leads to an earlier onset of polyploidy, and polyploid cells in the adult brain are more resistant to DNA damage-induced cell death than diploid cells. Our results suggest polyploidy may serve a protective role for neurons and glia in ageingDrosophila melanogasterbrains.

Summary In this issue of Developmental Cell, Cohen et al. show that the Drosophila hindgut is a genetically tractable model for studying tissue regeneration. This tissue exhibits different regeneration strategies at different developmental times, demonstrating that the hindgut developmental clock, not tissue type, dictates the mode and capacity for regeneration.

Purpose Crizotinib, a mesenchymal-epithelial transition/anaplastic lymphoma kinase/c-ros oncogene 1 (ROS1) inhibitor, has recently been approved by the US Food and Drug Administration for the treatment of patients with advanced ROS1-positive non–small-cell lung cancer (NSCLC). Therefore, interest in ROS1 testing is growing. ROS1 gene fusions affect approximately 0.5% to 2% of unselected NSCLCs. Limited data are available on the prevalence and distribution of ROS1 fusions in patients with advanced-stage NSCLC. Material and Methods A series of 727 lung adenocarcinomas from patients with stage IV disease, negative for epidermal growth factor receptor and anaplastic lymphoma kinase alterations, were tested for ROS1 fusions by fluorescent in situ hybridization analysis, with confirmation by immunohistochemistry. Results were correlated with clinicopathologic parameters and compared with data from the literature. Results ROS1 fusions were detected in 29 patients (4%), including 27 of 266 females (10.2%) and two of 461 males (0.4%; P = 1.2E-10). The mean age of patients with ROS1-positive disease was lower than that of patients with ROS1-negative disease (49.21 v 62.96 years, respectively; P = 1.1E-10). Eleven of 583 smokers (1.9%) and 18 of 144 nonsmokers (12.5%) showed ROS1 rearrangement ( P = 4.05E-7). By logistic regression analysis, ROS1 fusions were independently associated with female sex, younger age at diagnosis, and absence of smoking history, (odds ratios, 12.4, 7.9, and 3.6, respectively). These data, integrated with those reported in the literature, indicate that the prevalence of ROS1 fusions in females and in nonsmokers was higher in patients with advanced disease than in patients with operable disease (11.2% v 3.1%, P < .001; 11.6% v 2.8%, P < .001, respectively). The mean age at diagnosis was significantly lower in patients with advanced disease (49.8 years) than in patients with operable disease (55.6 years; P < .001). Conclusion Our data indicate that ROS1 fusions in patients with advanced-stage lung adenocarcinoma are more frequent in females, particularly if young and nonsmokers. A diagnostic algorithm for an accurate screening of ROS1 alterations was elaborated.

Background Genome organization changes during development as cells differentiate. Chromatin motion becomes increasingly constrained and heterochromatin clusters as cells become restricted in their developmental potential. These changes coincide with slowing of the cell cycle, which can also influence chromatin organization and dynamics. Terminal differentiation is often coupled with permanent exit from the cell cycle, and existing data suggest a close relationship between a repressive chromatin structure and silencing of the cell cycle in postmitotic cells. Heterochromatin clustering could also contribute to stable gene repression to maintain terminal differentiation or cell cycle exit, but whether clustering is initiated by differentiation, cell cycle changes, or both is unclear. Here we examine the relationship between chromatin organization, terminal differentiation and cell cycle exit. Results We focused our studies on the Drosophila wing, where epithelial cells transition from active proliferation to a postmitotic state in a temporally controlled manner. We find there are two stages of G0 in this tissue, a flexible G0 period where cells can be induced to reenter the cell cycle under specific genetic manipulations and a state we call “robust,” where cells become strongly refractory to cell cycle reentry. Compromising the flexible G0 by driving ectopic expression of cell cycle activators causes a global disruption of the clustering of heterochromatin-associated histone modifications such as H3K27 trimethylation and H3K9 trimethylation, as well as their associated repressors, Polycomb and heterochromatin protein 1 (HP1). However, this disruption is reversible. When cells enter a robust G0 state, even in the presence of ectopic cell cycle activity, clustering of heterochromatin-associated modifications is restored. If cell cycle exit is bypassed, cells in the wing continue to terminally differentiate, but heterochromatin clustering is severely disrupted. Heterochromatin-dependent gene silencing does not appear to be required for cell cycle exit, as compromising the H3K27 methyltransferase Enhancer of zeste, and/or HP1 cannot prevent the robust cell cycle exit, even in the face of normally oncogenic cell cycle activities. Conclusions Heterochromatin clustering during terminal differentiation is a consequence of cell cycle exit, rather than differentiation. Compromising heterochromatin-dependent gene silencing does not disrupt cell cycle exit. Electronic supplementary material The online version of this article (10.1186/s13072-017-0159-8) contains supplementary material, which is available to authorized users.

The Drosophila melanogaster male accessory gland is a functional analog of the mammalian prostate containing two secretory epithelial cell types, termed main and secondary cells. This tissue is responsible for making and secreting seminal fluid proteins and other molecules that contribute to successful reproduction. Here we show that similar to the mammalian prostate, this tissue grows with age and can undergo functional regeneration post-damage. We describe a post-eclosion endocycle required for gland maturation, that is dependent on juvenile hormone signaling. In addition, the differentiated binucleated main cells of the adult accessory gland continue to undergo rare endocycles throughout physiological aging to increase DNA content and cell size. Accessory gland cells remain poised to endocycle and upregulation of signals that promote endocycling and tissue growth are sufficient to trigger dramatic increases in cell ploidy. We provide evidence that the main cells of this tissue remain poised to enter the endocycle to assist in tissue regrowth after damage, providing a novel model for postmitotic regeneration in a binucleate tissue.

While cellular proliferation is fundamental to the development of all multicellular organisms, the slowing or stopping of proliferation at the right places and times is equally important for proper tissue and organ development. The non-cycling state of cellular quiescence or “G0” is relatively understudied compared to proliferation, given its prevalence in nature. It may seem that actively proliferating cells undergo a series of dynamic events, while quiescent cells are in a passive, static state. However, studies over the last 10-15 years suggest that quiescence may be more dynamic than previously thought and must also be actively regulated and maintained. This review focuses on recent advances in understanding quiescence or G0 and in particular, on observations about the proliferation-quiescence decision in cell lines, in tissues and during development. We also discuss novel, advanced molecular tools that are likely to enable the field to address outstanding, unresolved questions about cellular quiescence and its regulation.

During development, cell proliferation and differentiation must be tightly coordinated to ensure proper tissue morphogenesis. Because steroid hormones are central regulators of developmental timing, understanding the links between steroid hormone signaling and cell proliferation is crucial to understanding the molecular basis of morphogenesis. Here we examined the mechanism by which the steroid hormone ecdysone regulates the cell cycle in Drosophila. We find that a cell cycle arrest induced by ecdysone in Drosophila cell culture is analogous to a G2 cell cycle arrest observed in the early pupa wing. We show that in the wing, ecdysone signaling at the larva-to-puparium transition induces Broad which in turn represses the cdc25c phosphatase String. The repression of String generates a temporary G2 arrest that synchronizes the cell cycle in the wing epithelium during early pupa wing elongation and flattening. As ecdysone levels decline after the larva-to-puparium pulse during early metamorphosis, Broad expression plummets, allowing String to become re-activated, which promotes rapid G2/M progression and a subsequent synchronized final cell cycle in the wing. In this manner, pulses of ecdysone can both synchronize the final cell cycle and promote the coordinated acquisition of terminal differentiation characteristics in the wing., Summary: Pulsed ecdysone signaling remodels cell cycle dynamics, causing distinct primary and secondary cell cycle arrests in Drosophila cells, analogous to those observed in the wing during metamorphosis.

Objectives Anaplastic Lymphoma Kinase (ALK) gene rearrangements have been described in 3–5% of lung adenocarcinomas (ADC) and their identification is essential to select patients for treatment with ALK tyrosine kinase inhibitors. For several years, fluorescent in situ hybridization (FISH) has been considered as the only validated diagnostic assay. Currently, alternative methods are commercially available as diagnostic tests. Material and methods A series of 217 ADC comprising 196 consecutive resected tumors and 21 ALK FISH-positive cases from an independent series of 702 ADC were investigated. All specimens were screened by IHC (ALK-D5F3-CDx-Ventana), FISH (Vysis ALK Break-Apart-Abbott) and RT-PCR (ALK RGQ RT-PCR-Qiagen). Results were compared and discordant cases subjected to Next Generation Sequencing. Results Thirty-nine of 217 samples were positive by the ALK RGQ RT-PCR assay, using a threshold cycle (Ct) cut-off ≤35.9, as recommended. Of these positive samples, 14 were negative by IHC and 12 by FISH. ALK RGQ RT-PCR/FISH discordant cases were analyzed by the NGS assay with results concordant with FISH data. In order to obtain the maximum level of agreement between FISH and ALK RGQ RT-PCR data, we introduced a new scoring algorithm based on the ΔCt value. A ΔCt cut-off level ≤3.5 was used in a pilot series. Then the algorithm was tested on a completely independent validation series. By using the new scoring algorithm and FISH as reference standard, the sensitivity and the specificity of the ALK RGQ RT-PCR(ΔCt) assay were 100% and 100%, respectively. Conclusions Our results suggest that the ALK RGQ RT-PCR test could be useful in clinical practice as a complementary assay in multi-test diagnostic algorithms or even, if our data will be confirmed in independent studies, as a standalone or screening test for the selection of patients to be treated with ALK inhibitors.

Robust and synchronous repression of E2F-dependent gene expression is critical to the proper timing of cell cycle exit when cells transition to a postmitotic state. Previously NuA4 was suggested to act as a barrier to proliferation in Drosophila by repressing E2F-dependent gene expression. Here we show that NuA4 activity is required for proper cell cycle exit and the repression of cell cycle genes during the transition to a postmitotic state in vivo. However, the delay of cell cycle exit caused by compromising NuA4 is not due to additional proliferation or effects on E2F activity. Instead NuA4 inhibition results in slowed cell cycle progression through late S and G2 phases due to aberrant activation of an intrinsic p53-independent DNA damage response. A reduction in NuA4 function ultimately produces a paradoxical cell cycle gene expression program, where certain cell cycle genes become derepressed in cells that are delayed during the G2 phase of the final cell cycle. Bypassing the G2 delay when NuA4 is inhibited leads to abnormal mitoses and results in severe tissue defects. NuA4 physically and genetically interacts with components of the E2F complex termed Drosophila, Rbf, E2F and Myb/Multi-vulva class B (DREAM/MMB), and modulates a DREAM/MMB-dependent ectopic neuron phenotype in the posterior wing margin. However, this effect is also likely due to the cell cycle delay, as simply reducing Cdk1 is sufficient to generate a similar phenotype. Our work reveals that the major requirement for NuA4 in the cell cycle in vivo is to suppress an endogenous DNA damage response, which is required to coordinate proper S and G2 cell cycle progression with differentiation and cell cycle gene expression.

// Younghun Jung 1 , Ann M. Decker 1 , Jingcheng Wang 1 , Eunsohl Lee 1 , Lulia A. Kana 1 , Kenji Yumoto 1 , Frank C. Cackowski 1, 2 , James Rhee 1 , Peter Carmeliet 3, 4 , Laura Buttitta 5 , Todd M. Morgan 6 , Russell S. Taichman 1 1 Department of Periodontics and Oral Medicine, University of Michigan School of Dentistry, Ann Arbor, MI, USA 2 Department of Internal Medicine, Division of Hematology and Oncology, University of Michigan School of Medicine, Ann Arbor, MI, USA 3 Laboratory of Angiogenesis and Vascular Metabolism, Vesalius Research Center (VRC), VIB, K.U. Leuven, Belgium 4 Laboratory of Angiogenesis and Vascular Metabolism, Department of Oncology, K.U. Leuven, Belgium 5 Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI, USA 6 Department of Urology, University of Michigan School of Medicine, Ann Arbor, MI, USA Correspondence to: Russell S. Taichman, e-mail: rtaich@umich.edu Keywords: endogenous GAS6, Mer, prostate cancer, cancer stem cells, bone marrow Received: December 01, 2015 Accepted: March 07, 2016 Published: March 25, 2016 ABSTRACT GAS6 and its receptors (Tryo 3, Axl, Mer or “TAM”) are known to play a role in regulating tumor progression in a number of settings. Previously we have demonstrated that GAS6 signaling regulates invasion, proliferation, chemotherapy-induced apoptosis of prostate cancer (PCa) cells. We have also demonstrated that GAS6 secreted from osteoblasts in the bone marrow environment plays a critical role in establishing prostate tumor cell dormancy. Here we investigated the role that endogenous GAS6 and Mer receptor signaling plays in establishing prostate cancer stem cells in the bone marrow microenvironment. We first observed that high levels of endogenous GAS6 are expressed by disseminated tumor cells (DTCs) in the bone marrow, whereas relatively low levels of endogenous GAS6 are expressed in PCa tumors grown in a s.c. setting. Interestingly, elevated levels of endogenous GAS6 were identified in putative cancer stem cells (CSCs, CD133+/CD44+) compared to non-CSCs (CD133–/CD44–) isolated from PCa/osteoblast cocultures in vitro and in DTCs isolated from the bone marrow 24 hours after intracardiac injection. Moreover, we found that endogenous GAS6 expression is associated with Mer receptor expression in growth arrested (G 1 ) PCa cells, which correlates with the increase of the CSC populations. Importantly, we found that overexpression of GAS6 activates phosphorylation of Mer receptor signaling and subsequent induction of the CSC phenotype in vitro and in vivo . Together these data suggest that endogenous GAS6 and Mer receptor signaling contribute to the establishment of PCa CSCs in the bone marrow microenvironment, which may have important implications for targeting metastatic disease.

Several small molecules, epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs), such as gefitinib, erlotinib and afatinib, have been demonstrated to significantly improve clinical outcomes in patients with advanced EGFR-mutated non-small cell lung cancer (NSCLC), but erlotinib activity in EGFR wild-type squamous carcinoma is still highly debated. Here, we describe a prolonged and unexpected clinical response to erlotinib in a male former heavy cigarette smoker with wild-type EGFR squamous-cell cancer.

During terminal differentiation most cells will exit the cell cycle and enter into a prolonged or permanent G0. Cell cycle exit is usually initiated through the repression of cell cycle gene expression by formation of a transcriptional repressor complex called DREAM. However when DREAM repressive function is compromised during terminal differentiation, additional unknown mechanisms act to stably repress cycling and ensure robust cell cycle exit. Here we provide evidence that developmentally programmed, temporal changes in chromatin accessibility at a subset of critical cell cycle genes acts to enforce cell cycle exit during terminal differentiation in the Drosophila melanogaster wing. We show that during terminal differentiation, chromatin closes at a set of pupal wing enhancers for the key rate-limiting cell cycle regulators cycE, e2f1 and stg. This closing coincides with wing cells entering a robust postmitotic state that is strongly refractory to cell cycle re-activation. When cell cycle exit is genetically disrupted, chromatin accessibility at cell cycle genes remains largely unaffected and the closing of enhancers at cycE, e2f1 and stg proceeds independent of the cell cycling status. Instead, disruption of cell cycle exit leads to changes in accessibility and expression of a subset of hormone-induced transcription factors involved in the progression of terminal differentiation. Our results uncover a mechanism that acts as a cell cycle-independent timer to limit aberrant cycling in terminally differentiating tissues. In addition, we provide a new molecular description of the cross-talk between cell cycle exit and terminal differentiation during metamorphosis.

The sesquiterpene lactone parthenolide (PN) has recently attracted considerable attention because of its anti-microbial, anti-inflammatory and anticancer effects. However, the mechanism of its cytotoxic action on tumor cells remains scarcely defined. We recently provided evidence that the effect exerted by PN in MDA-MB-231 breast cancer cells was mediated by the production of reactive oxygen species (ROS). The present study shows that PN promoted the phosphorylation of EGF receptor (phospho-EGFR) at Tyr1173, an event which was observed already at 1 h of incubation with 25 µM PN and reached a peak at 8-16 h. This effect seemed to be a consequence of ROS production, because N-acetylcysteine (NAC), a powerful ROS scavenger, prevented the increment of phospho-EGFR levels. In addition fluorescence analyses performed using dihydroethidium demonstrated that PN stimulated the production of superoxide anion already at 2-3 h of incubation and the effect further increased prolonging the time of treatment, reaching a peak at 8-16 h. Superoxide anion production was markedly hampered by apocynin, a well known NADPH oxidase (NOX) inhibitor, suggesting that the effect was dependent on NOX activity. The finding that AG1478, an EGFR kinase inhibitor, substantially blocked both EGFR phosphorylation and superoxide anion production strongly suggested that phosphorylation of EGFR can be responsible for the activation of NOX with the consequent production of superoxide anion. Therefore, EGFR phosphorylation can exert a key role in the production of superoxide anion and ROS induced by PN in MDA-MB-231 cells.

IntroductionThe potential to accurately quantify epidermal growth factor receptor (EGFR) mutations in plasma from non–small-cell lung cancer patients would enable more rapid and more frequent analyses to assess disease status; however, the utility of such analyses for clinical purposes has only recently started to explore.MethodsPlasma samples were obtained from 69 patients with EGFR-mutated tumors and 21 negative control cases. EGFR mutations in plasma were analyzed by a standardized allele-specific polymerase chain reaction (PCR) test and ultra-deep next-generation sequencing (NGS). A semiquantitative index (SQI) was derived from dilutions of known EGFR mutation copy numbers. Clinical responses were evaluated by Response Evaluation Criteria in Solid Tumors 1.1 criteria and expressed as percent tumor shrinkage.ResultsThe sensitivity and specificity of the PCR test and NGS assay in plasma versus tissue were 72% versus 100% and 74% versus 100%, respectively. Quantitative indices by the PCR test and NGS were significantly correlated (p < 0.001). EGFR testing at baseline and serially at 4 to 60 days during tyrosine kinase inhibitor therapy revealed a progressive decrease in SQI, starting from day 4, in 95% of cases. The rate of SQI decrease correlated with percent tumor shrinkage at 2 months (p < 0.0001); at 14 days, it was more than 50% in 70% of patients (rapid responders). In two patients with slow response, an early increase in the circulating levels of the T790M mutation was observed. No early T790M mutations were seen in plasma samples of rapid responders.ConclusionsQuantification of EGFR mutations from plasma with a standardized PCR test is feasible. To our knowledge, this is the first study showing a strong correlation between the EGFR SQI in the first days of treatment and clinical response with relevant implications for patient management.

Resistance to RAF- and MEK-targeted therapy is a major clinical challenge1–4. RAF and MEK inhibitors are initially but only transiently effective in some but not all patients with BRAF gene mutation and are largely ineffective in those with RAS gene mutation because of resistance5–14. Through a genetic screen in BRAF-mutant tumor cells, we show that the Hippo pathway effector YAP (encoded by YAP1) acts as a parallel survival input to promote resistance to RAF and MEK inhibitor therapy. Combined YAP and RAF or MEK inhibition was synthetically lethal not only in several BRAF-mutant tumor types but also in RAS-mutant tumors. Increased YAP in tumors harboring BRAF V600E was a biomarker of worse initial response to RAF and MEK inhibition in patients, establishing the clinical relevance of our findings. Our data identify YAP as a new mechanism of resistance to RAF- and MEK-targeted therapy. The findings unveil the synthetic lethality of combined suppression of YAP and RAF or MEK as a promising strategy to enhance treatment response and patient survival.

Background and aim Adenosine triphosphate (ATP)-binding cassette (ABC) transporters A1 and G1 are the main transporters involved in macrophage cholesterol efflux. The understanding of the molecular mechanism(s) of their regulation in atherosclerosis is crucial for potential therapeutic approaches. Preclinical studies support a role for microRNAs in the posttranscriptional regulation of these transporters; however, no evidence is still available on human atherosclerosis. Thus, the aim of this study was to investigate the modulation of the ABCA1 and ABCG1 pathway in human atherosclerotic plaques and microRNA involvement in its modulation. Methods and results Thirty-one human atherosclerotic plaques were obtained from patients undergoing carotid endarterectomy for high-grade (>70%) vessel stenosis, and divided into normocholesterolemic (n = 15) and hypercholesterolemic groups (n = 16) according to the presence/absence of hypercholesterolemia. Both ABCA1 and ABCG1 messenger RNAs (mRNAs) were significantly upregulated in carotid plaques from hypercholesterolemic patients as assessed by real-time polymerase chain reaction (RT-PCR). Despite this result, no difference was found at the protein levels analyzed by Western blot, thus suggesting a strong posttranscriptional modulation. MicroRNA microarray and subsequent validation by RT-PCR showed a significant upregulation of ABCA1-linked miR-758 and miR-33b in plaques from hypercholesterolemic patients. Conclusion We provide evidence of a strong posttranscriptional regulation of ABCA1 and ABCG1 expression in human atherosclerotic plaques from hypercholesterolemic patients. This effect is potentially due to the concomitant increase of miR-33b and miR-758, two well-established regulators of ABCA1 and ABCG1 expression. The identification of miR-33b and miR-758 as putative key regulators of ABCA1 protein expression within human atherosclerotic plaques provides further data for the realization of new anti-atherosclerotic drugs with specific targets based on anti-miRNA technologies.

Type 2 diabetes is a chronic disease characterized by inadequate β-cell response to the progressive insulin resistance. MicroRNAs (miRNAs) are short, endogenous, noncoding RNAs representing a class of powerful gene expression modulators. Previous population studies observed a modulation of circulating miRNAs in diabetic patients; however, few data are presently available on miRNA modulation in diabetic patients naïve to pharmacological treatment as well as the effect of glycemic control on this.We aimed at studying circulating miRNA expression in diabetic patients naïve to treatment and at investigating the influence on this of glycemic control.This was a case-control study.Eighteen treatment-naïve diabetic patients with poor metabolic control and 12 control patients participated in the study.Wide miRNA expression profiling was performed, and the expression of miRNAs found to be dysregulated was then validated by quantitative RT-PCR. Finally, algorithm-identified putative miRNA targets were evaluated by quantitative RT-PCR and ELISA.In diabetic patients, microarray analysis showed that four miRNAs are increased, whereas 21 miRNAs are decreased. Quantitative RT-PCR validation confirmed the significant up-regulation of miR-326 (P = .004) and down-regulation of let-7a (P.001) and let-7f (P = .003). Notably, an inverse negative correlation was found between circulating miR-326 and its putative target adiponectin (p = -0.479, P = .009). After 12 months of antidiabetic treatment, quantitative RT-PCR data analysis showed that miR-326 levels were unaffected, whereas the levels of let-7a and let-7f were significantly increased.Treatment-naïve, poorly controlled diabetic patients show a significant dysregulation of miRNAs involved in the regulation of the adiponectin pathway, a phenomenon that may be reversed, at least in part, by improved glycemic control.

Cancer stem cells likely survive chemotherapy or radiotherapy by acquiring mutations that inactivate the endogenous apoptotic machinery or by cycling slowly. Thus, knowledge about the mechanisms linking the activation of an alternative cell death modality and the cell cycle machinery could have a transformative impact on the development of new cancer therapies, but the mechanisms remain completely unknown. We investigated the regulation of alternative cell death in Drosophila larval brain neural stem cells (neuroblasts) in which apoptosis is normally repressed. From a screen, we identified two novel loss-of-function alleles of the Cdc20/fizzy (fzy) gene that lead to premature brain neuroblast loss without perturbing cell proliferation in other diploid cell types. Fzy is an evolutionarily conserved regulator of anaphase promoting complex/cyclosome (APC/C). Neuroblasts carrying the novel fzy allele or exhibiting reduced APC/C function display hallmarks of necrosis. By contrast, neuroblasts overexpressing the non-degradable form of canonical APC/C substrates required for cell cycle progression undergo mitotic catastrophe. These data strongly suggest that Fzy can elicit a novel pro-survival function of APC/C by suppressing necrosis. Neuroblasts experiencing catastrophic cellular stress, or overexpressing p53, lose Fzy expression and undergo necrosis. Co-expression of fzy suppresses the death of these neuroblasts. Consequently, attenuation of the Fzy-dependent survival mechanism functions downstream of catastrophic cellular stress and p53 to eliminate neuroblasts by necrosis. Strategies that target the Fzy-dependent survival mechanism might lead to the discovery of new treatments or complement the pre-existing therapies to eliminate apoptosis-resistant cancer stem cells by necrosis.

Background: The Decapentaplegic (Dpp) signaling pathway is used in many developmental and homeostatic contexts, each time resulting in cellular responses particular to that biological niche. The flexibility of Dpp signaling is clearly evident in epithelial cells of the Drosophila wing imaginal disc. During larval stages of development, Dpp functions as a morphogen, patterning the wing developmental field and stimulating tissue growth. A short time later, however, as wing-epithelial cells exit the cell cycle and begin to differentiate, Dpp is a critical determinant of vein-cell fate. It is likely that the Dpp signaling pathway regulates different sets of target genes at these two developmental time points. Results: To identify mechanisms that temporally control the transcriptional output of Dpp signaling in this system, we have taken a gene expression profiling approach. We identified genes affected by Dpp signaling at late larval or early pupal developmental time points, thereby identifying patterning- and differentiation-specific downstream targets, respectively. Conclusions: Analysis of target genes and transcription factor binding sites associated with these groups of genes revealed potential mechanisms by which target-gene specificity of the Dpp signaling pathway is temporally regulated. In addition, this approach revealed novel mechanisms by which Dpp affects the cellular differentiation of wing-veins. Developmental Dynamics 243:818–832, 2014. © 2014 Wiley Periodicals, Inc.

During terminal differentiation, most cells exit the cell cycle and enter into a prolonged or permanent G0 in which they are refractory to mitogenic signals. Entry into G0 is usually initiated through the repression of cell cycle gene expression by formation of a transcriptional repressor complex called dimerization partner (DP), retinoblastoma (RB)-like, E2F and MuvB (DREAM). However, when DREAM repressive function is compromised during terminal differentiation, additional unknown mechanisms act to stably repress cycling and ensure robust cell cycle exit. Here, we provide evidence that developmentally programmed, temporal changes in chromatin accessibility at a small subset of critical cell cycle genes act to enforce cell cycle exit during terminal differentiation in the Drosophila melanogaster wing. We show that during terminal differentiation, chromatin closes at a set of pupal wing enhancers for the key rate-limiting cell cycle regulators Cyclin E (cycE), E2F transcription factor 1 (e2f1), and string (stg). This closing coincides with wing cells entering a robust postmitotic state that is strongly refractory to cell cycle reactivation, and the regions that close contain known binding sites for effectors of mitogenic signaling pathways such as Yorkie and Notch. When cell cycle exit is genetically disrupted, chromatin accessibility at cell cycle genes remains unaffected, and the closing of distal enhancers at cycE, e2f1, and stg proceeds independent of the cell cycling status. Instead, disruption of cell cycle exit leads to changes in accessibility and expression of a subset of hormone-induced transcription factors involved in the progression of terminal differentiation. Our results uncover a mechanism that acts as a cell cycle–independent timer to limit the response to mitogenic signaling and aberrant cycling in terminally differentiating tissues. In addition, we provide a new molecular description of the cross talk between cell cycle exit and terminal differentiation during metamorphosis., The longer a cell remains in G0, the more refractory it becomes to re-entering the cell cycle. This study shows that in terminally differentiated cells in vivo, regulatory elements at genes encoding just three key cell cycle regulators (cycE, e2f1 and stg) become inaccessible, limiting their aberrant activation and maintaining a prolonged, robust G0.

Chondrodysplasia punctata (CP) is a heterogeneous group of bone dysplasias that are characterized by abnormal calcium deposition in areas of enchondral bone formation. The existence of an X-linked recessive form of chondrodysplasia punctata (CDPX) has been recognized in patients who are nullisomic for the Xp22.3 region, presenting with complex phenotypes. The gene of CDPX has been identified recently, and five point mutations of the gene, named ARSE, have been described. Here, we report on the clinical and molecular characterization of a patient with CDPX. The patient presented at birth with cranial and facial anomalies and short stature; an x-ray skeletal survey showed punctate calcifications and striking hand and foot abnormalities. Single strand conformation polymorphism (SSCP) and sequence analysis of the patient's DNA allowed the identification of a new mutation of the ARSE gene; this mutation causes an amino acid substitution from cysteine to tyrosine at position 492 of the ARSE predicted protein product. The clinical description of patients with CDPX due to known mutation of the ARSE is of interest for the precise delineation of the clinical spectrum of the disease.

The sesquiterpene lactone Parthenolide (PN) exerted a cytotoxic effect on MDA-MB231 cells, a triple-negative breast cancer (TNBC) cell line, but its effectiveness was scarce when employed at low doses. This represents an obstacle for a therapeutic utilization of PN. In order to overcome this difficulty we associated to PN the suberoylanilide hydroxamic acid (SAHA), an histone deacetylase inhibitor. Our results show that SAHA synergistically sensitized MDA-MB231 cells to the cytotoxic effect of PN. It is noteworthy that treatment with PN alone stimulated the survival pathway Akt/mTOR and the consequent nuclear translocation of Nrf2, while treatment with SAHA alone induced autophagic activity. However, when the cells were treated with SAHA/PN combination, SAHA suppressed PN effect on Akt/mTOR/Nrf2 pathway, while PN reduced the prosurvival autophagic activity of SAHA. In addition SAHA/PN combination induced GSH depletion, fall in m, release of cytochrome c, activation of caspase 3 and apoptosis. Finally we demonstrated that combined treatment maintained both hyperacetylation of histones H3 and H4 induced by SAHA and down-regulation of DNMT1 expression induced by PN. Inhibition of the DNA-binding activity of NF-kB, which is determined by PN, was also observed after combined treatment. In conclusion, combination of PN to SAHA inhibits the cytoprotective responses induced by the single compounds, but does not alter the mechanisms leading to the cytotoxic effects. Taken together our results suggest that this combination could be a candidate for TNBC therapy. J. Cell. Physiol. 230: 1276-1289, 2015. (c) 2014 Wiley Periodicals, Inc., A Wiley Company

Specification of tissue identity during development requires precise coordination of gene expression in both space and time. Spatially, master regulatory transcription factors are required to control tissue-specific gene expression programs. However, the mechanisms controlling how tissue-specific gene expression changes over time are less well understood. Here, we show that hormone-induced transcription factors control temporal gene expression by regulating the accessibility of DNA regulatory elements. Using the Drosophila wing, we demonstrate that temporal changes in gene expression are accompanied by genome-wide changes in chromatin accessibility at temporal-specific enhancers. We also uncover a temporal cascade of transcription factors following a pulse of the steroid hormone ecdysone such that different times in wing development can be defined by distinct combinations of hormone-induced transcription factors. Finally, we show that the ecdysone-induced transcription factor E93 controls temporal identity by directly regulating chromatin accessibility across the genome. Notably, we found that E93 controls enhancer activity through three different modalities, including promoting accessibility of late-acting enhancers and decreasing accessibility of early-acting enhancers. Together, this work supports a model in which an extrinsic signal triggers an intrinsic transcription factor cascade that drives development forward in time through regulation of chromatin accessibility.

Background No target therapies are presently available in the treatment of small-cell lung cancer (SCLC). We investigated the presence of potentially drugable mutations in the EGFR, c-MET, BRAF, KRAS, PDGFRa and c-KIT genes in a retrospective series of SCLC from 2 Italian Institutions. Correlations with immunohistochemical, clinical and outcome features were evaluated. Materials and methods Genes were studied by direct sequencing of DNA extracted from formalin-fixed paraffin-embedded tissues. Immunohistochemical expression of TTF-1, p63, chromogranin, synaptophysin, CD56 and bcl-2 was assessed. Results Samples from 113 SCLC patients were analyzed. All cases were wild-type for BRAF, KRAS, PDGFRa and c-KIT (data available for 82 patients). Two (1.8%) patients were EGFR-mutated (exon 19 delE746-A750 and exon 21 L858R); both were females, non-smoker and had limited disease. Overall survival of EGFR-mutated patients was 21 months as compared to 11 months in wild-type. Five (4.4%) patients were c-MET-mutated (4 on exon 14: 2 R988C, 1 D990N, 1 D102Y; 1 on exon 17 R1166Q); all were smokers, 3 were males and 4 had extensive disease. Their OS was comparable to wild-type cases (12 vs. 11 months). EGFR and c-MET mutations were mutually exclusive. Gene mutations did not correlate with immunophenotype. Conclusions Targetable mutations are uncommon in SCLC. EGFR-mutated patients tended to be female and non-smoker and experienced a prolonged OS suggesting a possible positive prognostic effect. c-MET mutations did not affect survival. Target therapy might be considered in EGFR and c-MET-mutated patients.

Overexpression of both CycE and E2F is necessary to trigger cell cycle reentry and overproliferation of terminally differentiated wing cells., Terminally differentiated cells in Drosophila melanogaster wings and eyes are largely resistant to proliferation upon deregulation of either E2F or cyclin E (CycE), but exogenous expression of both factors together can bypass cell cycle exit. In this study, we show this is the result of cooperation of cell cycle control mechanisms that limit E2F-CycE positive feedback and prevent cycling after terminal differentiation. Aberrant CycE activity after differentiation leads to the degradation of E2F activator complexes, which increases the proportion of CycE-resistant E2F repressor complexes, resulting in stable E2F target gene repression. If E2F-dependent repression is lost after differentiation, high anaphase-promoting complex/cyclosome (APC/C) activity degrades key E2F targets to limit cell cycle reentry. Providing both CycE and E2F activities bypasses exit by simultaneously inhibiting the APC/C and inducing a group of E2F target genes essential for cell cycle reentry after differentiation. These mechanisms are essential for proper development, as evading them leads to tissue outgrowths composed of dividing but terminally differentiated cells.

Systematic sequence profiling of the Glioblastoma Multiforme (GBM) genome has recently led to the identification of somatic mutations in the isocitrate dehydrogenase 1 (IDH1) gene. Interestingly, only the evolutionarily conserved residue R132 located in the substrate binding site of IDH1 was found mutated in GBM. At present, the occurrence and the relevance of p.R132 (IDH1 R132) variants in tumors other than GBMs is largely unknown. We searched for mutations at position R132 of the IDH1 gene in a panel of 672 tumor samples. These included high-grade glioma, gastrointestinal stromal tumors (GIST), melanoma, bladder, breast, colorectal, lung, ovarian, pancreas, prostate, and thyroid carcinoma specimens. In addition, we assessed a panel of 84 cell lines from different tumor lineages. Somatic mutations affecting the IDH1R132 residue were detected in 20% (23 of 113) high-grade glioma samples. In addition to the previously reported p.R132H and p.R132S alleles, we identified three novel somatic mutations (p.R132C, p.R132G, and p.R132L) affecting residue IDH1R132 in GBM. Strikingly, no IDH1 mutations were detected in the other tumor types. These data indicate that cancer mutations affecting IDH1R132 are tissue-specific, and suggest that it plays a unique role in the development of high-grade gliomas.

It has been shown that the sesquiterpene lactone parthenolide lowers the viability of MDA-MB-231 breast cancer cells, in correlation with oxidative stress. The present report examined the different radical species produced during parthenolide treatment and their possible role in the toxicity caused by the drug. Time course experiments showed that in the first phase of treatment (0-8 h), and in particular in the first 3 h, parthenolide induced dichlorofluorescein (DCF) signal in a large percentage of cells, while dihydroethidium (DHE) signal was not stimulated. Since the effect on DCF signal was suppressed by apocynin and diphenyleneiodonium (DPI), two inhibitors of NADPH oxidase (NOX), we suggest that parthenolide rapidly stimulated NOX activity with production of superoxide anion (O2•-), which was converted by superoxide dismutase 1 (SOD1) into hydrogen peroxide (H2O2). In the second phase of treatment (8-16 h), parthenolide increased the number of positive cells to DHE signal. Since this event was not prevented by apocynin and DPI and was associated with positivity of cells to MitoSox Red, a fluorochrome used to detect mitochondrial production of O2•-, we suggest that parthenolide induced production of O2•- at the mitochondrial level independently by NOX activity in the second phase of treatment. Finally, in this phase, most cells became positive to hydroxyphenyl fluorescein (HPF) signal, a fluorescent probe to detect highly reactive oxygen species (hROS), such as hydroxyl radical and peroxynitrite. Therefore, parthenolide between 8-16 h of treatment induced generation of O2•- and hROS, in close correlation with a marked reduction in cell viability.

The complexity and plasticity of cell fate determination has intrigued cell and developmental biologists for decades. Cellular differentiation is the acquisition of specialized characteristics; which is intimately associated with changes in gene expression, alterations of chromatin, and changes in nuclear architecture. Differentiating tissues exhibit a progressive restriction of cellular plasticity. However, the regenerative ability of some organisms has revealed an amazing capacity for dramatic switches in cell fate, through trans-differentiation and de-differentiation (Sanchez Alvarado and Tsonis, 2006). Furthermore, the groundbreaking work on somatic cell nuclear reprogramming and induced pluripotency has revealed that commitment to cell fate can be far more flexible than previously thought (Lensch and Mummery, 2013). In this research topic on cell fate we aimed to highlight new developments and outstanding questions in our understanding of how chromatin dynamics impact cell fate and cellular reprogramming. We include articles discussing cell fate decisions in a wide variety of contexts and model organisms. The contributions to this topic include review articles, mini-reviews, original research, and perspectives. The work described here encompasses organisms ranging from C. elegans to humans and deals with global cell fate issues of sex determination (Lau and Csankovszki), lineage choice (Chin), preventing premature differentiation (Foret et al.) cell fate and cell cycle regulation (Oyama et al.; Julian and Blais; Ma et al.; Parker), nuclear architecture (Talamas and Capelson) and how dynamic transcriptional repressors promote cell fate choices (Kok and Arnosti). We thank the authors, reviewers and editors for contributing to the stimulating discussion of the open questions in this rapidly changing field.

The miR-200 microRNA family plays important tumor suppressive roles. The sole Drosophila miR-200 ortholog, miR-8 plays conserved roles in Wingless, Notch and Insulin signaling - pathways linked to tumorigenesis, yet homozygous null animals are viable and often appear morphologically normal. We observed that wing tissues mosaic for miR-8 levels by genetic loss or gain of function exhibited patterns of cell death consistent with a role for miR-8 in modulating cell survival in vivo. Here we show that miR-8 levels impact several actin cytoskeletal regulators that can affect cell survival and epithelial organization. We show that loss of miR-8 can confer resistance to apoptosis independent of an epithelial to mesenchymal transition while the persistence of cells expressing high levels of miR-8 in the wing epithelium leads to increased JNK signaling, aberrant expression of extracellular matrix remodeling proteins and disruption of proper wing epithelial organization. Altogether our results suggest that very low as well as very high levels of miR-8 can contribute to hallmarks associated with cancer, suggesting approaches to increase miR-200 microRNAs in cancer treatment should be moderate.

Activating mutations in the tyrosine kinase domain of the HER2 gene have recently been reported in lung adenocarcinomas, mainly in East Asian patients. Our study was devised to evaluate the prevalence and nature of HER2 mutations in lung adenocarcinomas from Caucasian patients. The mutational status of the HER2 gene was evaluated in 403 lung adenocarcinomas by PCR-single strand conformation polymorphism analysis and direct sequencing of Exons 19 and 20. We found HER2 mutations in 9 (2.2%) cases. Seven (78%) of the mutations were in frame duplications/insertions at codons 776-779 (YVMA), the other 2 were base substitutions resulting in aminoacid changes. The hotspot mutation at bases 776-779 was previously found to be the most frequent HER2 mutation in Asiatic patients. The distribution of mutations was significantly different between conventional lung adenocarcinomas (CLAs) and lung adenocarcinomas with bronchioloalveolar features (ABAFs). Seven (6.2%) of 113 ABAFs and 2 (0.7%) of 290 CLA were mutated (p = 0.0025). In addition, the frequency of HER2 mutations was slightly higher in females (4.1%) than in males (1.8%) and in never smokers (3.1%) than in smokers (1.9%), but differences were not statistically significant. This series of tumors was also investigated for EGFR and K-ras mutations. EGFR mutations were observed in 43 (10.7%) cases, and K-ras mutations in 110 (27.3%) cases. EGFR, HER2 and K-ras mutations were found to be mutually exclusive events. The presence of HER2 mutations in a subset of patients with lung adenocarcinoma raise hope to treat these patients with HER2 specific kinase inhibitors.

Objective— We recently demonstrated that inducible cyclooxygenase/PGE synthase-1 (COX-2/mPGES-1) are overexpressed in symptomatic plaques in association with PGE 2 -dependent metalloproteinase (matrix metalloproteinase [MMP]) biosynthesis and plaque rupture. However, it is not known which of the 4 PGE 2 receptors (EP1–4) mediates macrophage metalloproteinase generation. The aim of this study was to characterize EP1–4 expression in plaques from symptomatic and asymptomatic patients undergoing carotid endarterectomy and correlate it with the extent of inflammatory infiltration, COX-2/mPGES-1 and MMP expression and clinical features of patients’ presentation. Methods and Results— Plaques were analyzed for COX-2, mPGES-1, EP1–4, MMP-2, and MMP-9 by immunohistochemistry, reverse-transcription polymerase chain reaction and Western blot; zymography was used to detect MMP activity. We observed strong EP4 immunoreactivity, only very weak staining for EP2, and no expression of EP1 and EP3 in atherosclerotic plaques. EP4 was more abundant in MMP-rich symptomatic lesions, whereas EP2 was no different between symptomatic and asymptomatic plaques. Finally, MMP induction by PGE 2 in vitro was inhibited by the EP4 antagonist L-161 982, but not by its inactive analog L-161 983 or by the EP2 antagonist AH6809. Conclusions— This study shows that EP4 overexpression is associated with enhanced inflammatory reaction in atherosclerotic plaques. This effect might contribute to plaque destabilization by inducing culprit metalloproteinase expression.

Purpose: The Int6 gene was originally identified as a common insertion site for the mouse mammary tumor virus in virally induced mouse mammary tumors. Recent studies indicate that Int6 is a multifaceted protein involved in the regulation of protein translation and degradation through binding with three complexes: the eukaryotic translation initiation factor 3, the proteasome regulatory lid, and the constitutive photomorphogenesis 9 signalosome. This study aimed to investigate the prognostic role of Int6 in a large series of stage I non–small cell lung cancers (NSCLC) patients with long-term follow-up. Experimental Design: We determined the methylation status of Int6 DNA by methylation-specific PCR and the steady-state levels of Int6 RNA by quantitative real-time reverse transcription-PCR in 101 NSCLCs and matched normal lung tissues. Results: In 27% of the tumors, Int6 RNA levels were reduced relative to normal tissue. In 85% of the tumors with reduced Int6 expression, the transcription promoter and first exon were hypermethylated, whereas only 4% of the tumors with elevated Int6 RNA levels were hypermethylated (P < 0.000001). Low levels of Int6 RNA were found a significant predictor of overall and disease-free survival (P = 0.0004 and P = 0.0020, respectively). A multivariate analysis confirmed that low Int6 expression was the only independent factor to predict poor prognosis, for both overall (P = 0.0006) and disease-free (P = 0.024) survival. Conclusions: Our results suggest that Int6 expression, evaluated by quantitative real-time PCR, may represent a new prognostic factor in patients with stage I NSCLC.

Purpose It has been reported that EGFR mutations in lung carcinomas make the disease more responsive to treatment with tyrosine kinase inhibitors. We decided to evaluate the prevalence of EGFR mutations in a large series of non–small-cell lung carcinomas (NSCLCs) and to develop a rapid and sensitive screening method. Patients and Methods We examined 860 consecutive NSCLC patients for EGFR mutations in exons 18, 19, and 21 using a dual technical approach—direct sequencing of polymerase chain reaction (PCR) products and PCR single-strand conformation polymorphism (SSCP) analysis. Moreover, all lung adenocarcinomas were analyzed for K-ras mutations at codon 12 by allele-specific oligoprobe hybriditations. Results There were no EGFR mutations in 454 squamous carcinomas and 31 large cell carcinomas investigated. Thirty-nine mutations were found in the series of 375 adenocarcinomas (10%). Mutations were present in 26% of 86 bronchioloalveolar carcinomas (BACs) and in 6% of 289 conventional lung adenocarcinomas; P = .000002. EGFR mutations and K-ras mutations were mutually exclusive. A multivariable analysis revealed that BAC histotype, being a never smoker, and female sex were independently associated with EGFR mutations (odds ratios: 4.542, 3.632, and 2.895, respectively). The SSCP analysis was accurate and sensitive, allowing identification of mutations that were undetectable (21% of cases) by direct sequencing. Conclusion Mutations in the EGFR tyrosine kinase domain define a new molecular type of lung carcinoma, more frequent in particular subsets of patients. The SSCP assay is a rapid and reliable method for the detection of EGFR kinase domain mutations in lung cancer.

Sonic hedgehog (Shh) signaling is essential for sclerotome development in the mouse. Gli2 and Gli3 are thought to be the primary transcriptional mediators of Shh signaling; however, their roles in Shh induction of sclerotomal genes have not been investigated. Using a combination of mutant analysis and in vitro explant assays, we demonstrate that Gli2 and Gli3 are required for Shh-dependent sclerotome induction. Gli2 –/– Gli3 –/– embryos exhibit a severe loss of sclerotomal gene expression, and somitic mesoderm from these embryos cannot activate sclerotomal genes in response to exogenous Shh. We find that one copy of either Gli2 or Gli3 is required to mediate Shh induction of sclerotomal markers Pax1 and Pax9 in vivo and in vitro. Although Gli2 is generally considered an activator and Gli3 a repressor, our results also reveal a repressor function for Gli2 and an activator function for Gli3 in the developing somite. To further dissect the function of each Gli, we used adenovirus to overexpress Gli1, Gli2 and Gli3 in presomitic mesoderm explants. We find that each Gli preferentially activates a distinct set of Shh target genes, suggesting that the functions of Shh in patterning, growth and negative feedback are divided preferentially between different Gli proteins in the somite.

Survivin is an inhibitor of apoptosis protein, overexpressed in most human malignancies and implicated in mitosis regulation and preservation of cell viability. In order to investigate the prevalence and clinical significance of survivin in early-stage non-small cell lung carcinoma (NSCLC), survivin mRNA levels and protein expression were evaluated, using quantitative real-time RT-PCR and immunohistochemistry, respectively, in a series of 83 patients with stage I (IA and IB) surgically resected NSCLC. Detectable survivin mRNA levels could be demonstrated in all non-neoplastic lung tissue samples and in the tumours analysed. Survivin mRNA levels were elevated in 80 carcinomas (96%) compared to normal lung (p = 0.008). Among all tumours, survivin transcripts were present at a higher level in squamous cell carcinomas (p = 0.0022). Cytoplasmic and nuclear immunoreactivity was found in 70% and 80% of tumours, respectively and both were present in 54%. Cytoplasmic immunoreactivity correlated with tumour stage (p = 0.019). Survivin expression levels did not correlate with patient survival. In one specimen, cytoplasmic and focal nuclear immunostaining was observed in dysplastic bronchial squamous metaplasia. These results document that survivin overexpression is almost always present in early-stage NSCLC, suggesting that this protein may play a role in lung tumourigenesis. This ubiquitous expression makes survivin an appealing new target for novel therapies in lung cancer. In addition, this study also documents that survivin overexpression could be exploited for diagnostic purposes and that quantitative real-time RT-PCR can be a useful tool for evaluating survivin activation in NSCLC.

Since the earliest observations of cells undergoing mitosis, it has been clear that there is an intimate relationship between the cell cycle and nuclear chromatin architecture. The nuclear envelope and chromatin undergo robust assembly and disassembly during the cell cycle, and transcriptional and post-transcriptional regulation of histone biogenesis and chromatin modification is controlled in a cell cycle-dependent manner. Chromatin binding proteins and chromatin modifications in turn influence the expression of critical cell cycle regulators, the accessibility of origins for DNA replication, DNA repair, and cell fate. In this review we aim to provide an integrated discussion of how the cell cycle machinery impacts nuclear architecture and vice-versa. We highlight recent advances in understanding cell cycle-dependent histone biogenesis and histone modification deposition, how cell cycle regulators control histone modifier activities, the contribution of chromatin modifications to origin firing for DNA replication, and newly identified roles for nucleoporins in regulating cell cycle gene expression, gene expression memory and differentiation. We close with a discussion of how cell cycle status may impact chromatin to influence cell fate decisions, under normal contexts of differentiation as well as in instances of cell fate re-programming.

Protein phosphatase type 2A complex (PP2A) has been known as a tumor suppressor for over two decades, but it remains unclear exactly how it suppresses tumor growth. Here we provide data indicating a novel role for PP2A in promoting the transition to quiescence upon terminal differentiation in vivo. Using Drosophila eyes and wings as a model, we find that compromising PP2A activity during the final cell cycle prior to a developmentally controlled cell cycle exit leads to extra cell divisions and delayed entry into quiescence. By systematically testing the regulatory subunits of Drosophila PP2A, we find that the B56 family member widerborst (wdb) is required for the role of PP2A in promoting the transition to quiescence. Cells in differentiating tissues with compromised PP2A retain high Cdk2 activity when they should be quiescent, and genetic epistasis tests demonstrate that ectopic CyclinE/Cdk2 activity is responsible for the extra cell cycles caused by PP2A inhibition. The loss of wdb/PP2A function cooperates with aberrantly high Cyclin E protein, allowing cells to bypass a robust G0 late in development. This provides an example of how loss of PP2A can cooperate with oncogenic mutations in cancer. We propose that the wdb/PP2A complex plays a novel role in differentiating tissues to promote developmentally controlled quiescence through the regulation of CyclinE/Cdk2 activity.

Telomerase activity and telomerase reverse transcriptase (hTERT) expression are elevated in human malignancies. We have investigated telomerase activity measured by the telomeric repeat amplification protocol (TRAP) assay and hTERT levels by real-time RT-PCR in stage I non-small-cell lung carcinomas. The purposes of our study included the comparison of these two techniques in the assessment of telomerase function and the evaluation of their prognostic significance. Telomerase activity and hTERT levels were determined in 90 stage I non-small-cell lung carcinoma patients, using TRAP assay and real-time RT-PCR, respectively. Variables were analyzed by the chi(2) and Fisher exact tests. Survival was analyzed by the Kaplan-Meier method. Multivariate analysis was performed with the Cox's proportional hazards model. Telomerase activity was elevated in 60 (67%) carcinomas. hTERT was elevated in 43 (48%) carcinomas. Only 21 (23%) tumors had low telomerase function by both TRAP and hTERT expression levels. Telomerase activity and hTERT were significantly correlated (p = 0.017), although 35 cases displayed discordant results. Both telomerase activity and hTERT levels were significantly associated with poor patient overall and disease-free survival (p = 0.019 and p = 0.018 for TRAP, and p = 0.011 and p = 0.012 for hTERT, respectively). Among the 21 patients with tumors displaying low telomerase function, defined by both TRAP and hTERT expression levels, only one succumbed to the disease (p = 0.0053). Our results suggest that the two techniques used in this study evaluate separate aspects of telomerase function and their combination provides powerful prognostic information in lung cancer patients.

Dorsoventral polarity of the somitic mesoderm is established by competitive signals originating from adjacent tissues. The ventrally located notochord provides the ventralizing signals to specify the sclerotome, while the dorsally located surface ectoderm and dorsal neural tube provide the dorsalizing signals to specify the dermomyotome. Noggin and SHH-N have been implicated as the ventralizing signals produced by the notochord. Members of the WNT family of proteins, on the other hand, have been implicated as the dorsalizing signals derived from the ectoderm and dorsal neural tube. When presomitic explants are confronted with cells secreting SHH-N and WNT1 simultaneously, competition to specify the sclerotome and dermomyotome domains within the naive mesoderm can be observed. Here, using these explant cultures, we provide evidence that SHH-N competes with WNT1, not only by upregulating its own receptor Ptc1, but also by upregulating Sfrp2 (Secreted frizzled-related protein 2), which encodes a potential WNT antagonist. Among the four known Sfrps, Sfrp2 is the only member expressed in the sclerotome and upregulated by SHH-N recombinant protein. We further show that SFRP2-expressing cells can reduce the dermomyotome-inducing activity of WNT1 and WNT4, but not that of WNT3a. Together, our results support the model that SHH-N at least in part employs SFRP2 to reduce WNT1/4 activity in the somitic mesoderm.

How do developing organs sense and limit their size? The recently discovered Hippo pathway might have a critical role in controlling organ size and homeostasis in many organisms from Drosophila to mammals.

Fifty-two sporadic primary non-small-cell lung carcinomas (NSCLC) were examined for microsatellite instability. Six different microsatellite markers localized on chromosomes 2, 5, 8, 10, 11 and 17 were used. Genomic instability was observed in 35% (18/52) of NSCLC at single or multiple loci. The tumors were also analyzed for p53-gene mutations by PCR-SSCP analysis. Polynucleotide stretch frameshift mutations of TGFβRII (transforming-growth-factor-beta receptor II), IGFIIR (insuline growth-factor II receptor) and BAX genes were also analyzed. RER+ (replication-error-positive) tumors appear not to be affected by a higher rate of point mutations in coding sequences: no correlation was found between microsatellite instability and point mutations in the p53 gene, and the RER+ tumors showed no alterations in stretches of nucleotide inside TGFβRII, BAX or IGFIIR. Int. J. Cancer 78:606–609, 1998. © 1998 Wiley-Liss, Inc.

Lung cancer is strictly associated with tobacco smoking. Tumours developed in non-smoking subjects account for less than 10% of all lung cancers and show peculiar histopathological features, being prevalently adenocarcinomas. A number of genetic data suggest that their biological behaviour may be different from that of lung tumours caused by smoking, however the number of cases investigated to date is too low to draw definitive conclusions. We have examined the status of p53 and K-ras genes and the presence of loss of heterozygosity (LOH) at the FHIT locus in a series of 35 lung adenocarcinomas that developed in subjects who had never smoked. Results were compared with those obtained in a series of 35 lung adenocarcinomas from heavy-smoking subjects. In the group of non-smoking subjects p53 mutations and LOH at the FHIT locus were present in seven (20%) cases, and the two alterations were constantly associated (P < 0.0001), whereas they were not related in the series of carcinomas caused by smoking. In tumours developed in heavy-smoking subjects, the frequency of LOH at the FHIT locus was significantly higher (P = 0.006) than in tumours from non-smoking subjects. The frequency of p53 mutations in adenocarcinomas caused by smoking was not different from that seen in non-smoking subjects. However, in the group of smoking subjects we observed mostly G:C --> T:A transversions, whereas frameshift mutations and G:C --> A:T transitions were more frequently found in tumours from non-smoking subjects. No point mutations of the K-ras gene at codon 12 were seen in subjects who had never smoked, whereas they were present (mostly G:C --> T:A transversions) in 34% of tumours caused by smoking (P = 0.002). Our data suggest that lung adenocarcinomas developed in subjects who had never smoked represent a distinct biological entity involving a co-alteration of the p53 gene and the FHIT locus in 20% of cases.

Bronchioloalveolar carcinoma (BAC) is a particular type of adenocarcinoma of the lung which accounts for up to 9 per cent of pulmonary malignancies. The aetiology and pathogenesis of this unique neoplastic disease are still unclear. Three histological subtypes of BAC have been recognized: mucinous, non-mucinous, and sclerosing. Of these, mucinous and sclerosing BAC have a worse prognosis than non-mucinous tumours. The different morphological patterns and clinical outcomes of the subtypes of BAC suggest differences in their biological behaviour. Previous reports have shown that the mucinous form of BAC is characterized by constant mutations at codon 12 of the K-ras gene, whereas the other two histotypes show a frequency of K-ras mutations which is not different from that observed in conventional lung adenocarcinomas. The present study of a series of 51 BACs, previously investigated for K-ras gene mutations, has evaluated the status of two other genes, p53 and FHIT, known to be frequently altered in non-small cell lung cancer. Loss of heterozygosity at microsatellite-containing loci located within the FHIT gene was observed in 22 (43 per cent) BACs. The distribution of FHIT gene abnormalities was not statistically different in the three histological subtypes. p53 mutations were present in 13 (32 per cent) non-mucinous/sclerosing BACs, while no mutations were seen in mucinous tumours (P-0·039). Correlations with clinicopathological parameters showed that p53 mutations in BACs are associated with more aggressive tumours. No correlations were observed between FHIT or K-ras gene abnormalities and clinicopathological data. In conclusion, these results indicate that FHIT alterations are frequently involved in BAC tumourigenesis and that genetic changes in the p53 and K-ras genes can distinguish between different histotypes of BAC. © 1998 John Wiley & Sons, Ltd.