Category: clinically Small Molecule

We used a complementary gene expression profiling using the DNA microarray technology to monitor cellular changes in gene expression and discover the molecular targets upon RUNX2 suppression in EOC cells. To our knowledge, the present work represents the first effort to define global changes in gene expression upon modulation of RUNX2 expression in cancer cells. We analyzed both functionally related genes that were commonly differentially expressed in SKOV3 EOC cells upon RUNX2 knockdown. The gene expression data and consecutive network and pathway analyses were quite confirmatory of the data obtained by the RUNX2 functional assays. Indeed, microarray data sustained RUNX1 correlation with EOC cell proliferation, migration and invasion, since RUNX2 knockdown resulted in reduced expression of genes associated with metabolism, cellular growth & proliferation and cellular movement, while a number of genes linked to cell death were induced. IPA network analysis was indicative for some important gene nodes linked to RUNX2 suppression in EOC cells, as most of these substantiate and/or complement the functional data obtained. Thus, RUNX2 knockdown resulted in (+)-JQ1 upregulation of gene nodes/genes known to be implicated in apoptosis induction or displaying TSG functions. Notably the UBC interaction network and its members were shown to decrease in anchorage-independent cell growth and increase apoptosis, suggesting UBC may act as a negative regulator of skin carcinogenesis; CRYAB has been reported as a potential TSG, while increased expression of BCL-XS protein in tumors was associated with decreased proliferation and induction of apoptosis. Similarly, CTGF upregulation was found to be associated with apoptosis and decrease of tumor cell invasion; PPM1A expression could induce cell cycle arrest and apoptosis via activation of the p53 pathway, and NF2 has been characterized as a TSG in different cancers. Given the similar roles of RUNX1 and RUNX2 in EOC progression and the fact that all three RUNX proteins recognize common DNA sequence motifs, we analyzed the extent of overlap in differentially expressed genes/functional pathways following RUNX1 and RUNX2 knockdown in the SKOV3 ovarian adenocarcinoma cell line. Both the Venn diagram comparisons, as well as gene clustering and IPA functional analyses were indicative for distinct molecular mechanisms and functional pathways associated with RUNX1 or RUNX2 implication in EOC progression, although both genes could potentially modulate the expression of some common genes involved in EOC disease advancement and metastasis. In conclusion, we have shown that the RUNX2 transcription factor is significantly overexpressed in serous EOC tumors, including LMP tumors, compared to normal ovarian tissue. BSP validation of the RUNX2 methylation status in primary EOC tumors and omental metastasis were indicative for no implication of LY2835219 epigenetics mechanisms in RUNX2 overexpression in metastatic tissues. Further functional analyses of RUNX2 in EOC cells pointed towards its association with EOC cell proliferation, migration and invasion. Gene expression profiling and consecutive network and pathway analyses confirmed these findings, as various genes and pathways known previously to be implicated in ovarian tumorigenesis, including EOC tumor invasion and metastasis, were found to be suppressed upon RUNX2 knockdown, while a number of pro-apoptotic genes and some EOC TSGs were found to be induced. Our data suggest that RUNX2 is possibly implicated in EOC tumor and cancer cell growth and invasion and could represent a potential EOC therapeutic target. The present study also reveals that RUNX1 and RUNX2 employ distinct molecular mechanisms in EOC tumorigenesis despite evident similarities of their action on EOC cell phenotype and behavior.

In summary, our data suggest that dopaminergic function is differentially altered in mesolimbic and nigrostriatal MK-0683 pathways in the Fmr1-/Y mouse model of FXS, the AG-013736 319460-85-0 balance of effects facilitating reward function and diminishing motor function. While the importance of cholinergic actions in the striatum is appreciated, and anticholinergics have been in routine clinical use for extrapyramidal movement disorders for decades, the function of large, aspiny, tonically active cholinergic interneurons remains much less understood than other striatal cell types, such as MSNs and GABAergic fast-spiking interneurons, particularly in the NAc. TANs synapse extensively with MSNs and are the primary source of acetylcholine in both the dorsal striatum and NAc. As is the case for their role in motor function, the euphoric and rewarding effects of muscarinic anticholinergics have been appreciated for centuries, but the exact mechanisms by which anticholinergics function in mesolimbic brain reward circuitry are not well understood. Recent studies have suggested that TANs may integrate thalamostriatal and corticostriatal input and local FSI function to regulate firing of MSNs. Most of what is known regarding TAN function has been learned from experiments in the dorsal striatum, and with a few exceptions these neurons have been largely uninvestigated in the NAc. The net behavioral effects of the M1 antagonist, trihexyphenidyl, in WT mice were potentiation of BSR within a narrow dose range, suppression of maximum operant response rate without a change in threshold at the highest dose tested, and locomotor stimulation at a dose that potentiated BSR. In contrast, trihexyphenidyl did not potentiate BSR in Fmr1-/Y mice at any dose, and the locomotor stimulation we observed at 10 mg/kg was significantly enhanced in Fmr1-/Y compared to WT mice, consistent with a parkinsonian motor phenotype and further illustrating the dissociation of reward and motor effects in Fmr1-/Y mice. Trihexyphenidyl has no effect by itself on dopamine release in the NAc, but increases cocaine-potentiated NAc dopamine release and cocaine-stimulated locomotor activation, suggesting that cholinergic regulation of mesolimbic circuitry normally keeping NAc dopamine release in check is disinhibited by M1 antagonism, and that this function may be impaired in Fmr1-/Y mice. Acetylcholine signaling through M2/M4 receptors on TANs indirectly reduces dopamine release, but signaling through M1 receptors on presynaptic terminals increases excitability of dopaminergic neurons by inhibiting local GABA release in the VTA. Neither mechanism fully explains our present behavioral results with trihexyphenidyl. We are currently investigating how M1 activity in NAc MSNs differs between Fmr1-/Y and WT mice. Given that M1 receptors are also Gq-coupled and signal through PLC, it is possible that M1 function is also altered in NAc MSNs of mice lacking FMRP. Group I mGluR receptors are distributed ubiquitously throughout the brain and act as a brake on glutamatergic excitation. The highly selective mGluR5 antagonist, MPEP, has anxiolytic potency in rodent models and potentiated BSR in our hands, with larger effects in Fmr1-/Y than in WT mice. MPEP also transiently but significantly stimulated locomotor activity, although relative motor stimulation compared to baseline activity was similar between genotypes. In the dorsal striatum, mGluR5 receptors mediate both postsynaptic AMPA receptor endocytosis, which is enhanced in hippocampal neurons of Fmr1-/Y mice, and presynaptic endocannabinoid -dependent LTD. It has been shown that mGluR5-coupled retrograde eCB signaling through Gq-coupled activation of PLC and diacylglycerol lipase is disrupted in both dorsal striatum and NAc, and, in contrast to hippocampus, mGluR5-dependent LTD is absent from NAc MSNs in Fmr1-/Y mice.

The dopamine, glutamate, and acetylcholine systems in the brain are all affected in mice lacking Fmr1. Dopamine in particular is important for the initiation and reinforcement of motivated behaviors. Mice lacking Fmr1 have increased dopamine turnover but decreased amphetaminestimulated dopamine release in the dorsal striatum, which correlates with decreased sensitivity to amphetamine-induced motor stereotypies; as well as increased dopamine release in the prefrontal cortex. The postsynaptic effects of dopamine D1 receptor activity on AMPA-type glutamate receptor function are also reduced in both prefrontal cortex and striatum. There are relatively fewer behavioral or neurochemical studies on limbic motor system function in Fmr1-null mice than in hippocampus or neocortex. Given the critical involvement of limbic circuitry in motivation and reinforcement, changes in social integrative behavior and motor learning in FXS may be affected by underlying deficits in limbic brain reward circuitry as well as in cortex involved in memory and higher cognitive functions. Although two studies have shown normal acquisition of operant behavior using sucrose or food reinforcement in Fmr1-null mice, the neural mechanisms underlying motivation and reward have not been explored in depth in this model. Imaging studies have identified alterations in both morphology and activation patterns in the 3,4,5-Trimethoxyphenylacetic acid striatum of FXS patients, but the function of dopaminergic projections from the midbrain substantia nigra pars compacta and ventral tegmental area to their forebrain targets in the dorsal striatum and nucleus accumbens have been less extensively investigated than cortical circuits in Fmr1-null mice. Drugs that directly affect the dopamine system, including atypical neuroleptics such as aripiprazole, are of interest for the management of affective and behavioral symptoms in FXS. Cholinergic mechanisms in mesolimbic and nigrostriatal motor function, in which interactions with the dopamine system shape striatal output, are largely unexplored in this model. The goal of the current study was to characterize limbic motor circuitry with behavioral and neurochemical methods in Fmr1-null mice. Intracranial self-stimulation is an operant behavior in which animals perform a task for reinforcement by electrical brain stimulation reward. The predictable effects on BSR of drugs acting through dopamine, glutamate, or acetylcholine receptors can be compared between genotypes, and we have previously used this approach to investigate pharmacological mechanisms in other monogenic neurodevelopmental disorders. We hypothesize that Fmr1-null mice will show increased sensitivity to drugs that enhance the rewarding value of BSR and, conversely, decreased sensitivity to the reward-devaluing effects of drugs that diminish BSR. Experiments measuring the effects of the atypical neuroleptic aripiprazole, the mGluR5 antagonist MPEP, and the preferential M1 antagonist trihexyphenidyl on locomotor behavior were also performed to further differentiate drug effects on global motor function from effects specific to operant behavior. To LOUREIRIN-B determine if absence of Fmr1 alters dopaminergic neurons originating in the SNc and VTA, tyrosine hydroxylase immunoreactivity was also quantified by design-based stereology in midbrain histological sections and by western blot in tissue homogenates from dorsal striatum and NAc. We also measured the numbers of neurons expressing tyrosine hydroxylase, the rate-limiting enzyme, in the midbrain, and measured TH expression in forebrain targets of projections from those neurons. Our data suggest that absence of Fmr1 does not affect intrinsic sensitivity of mesolimbic circuits to brain stimulation reward, which we have previously shown to be increased in mice lacking the maternal allele of ubiquitin ligase, a model for Angelman syndrome.

Similar to RUNX1, the strongest evidence for a pro-oncogenic function for RUNX2 comes from studies in lymphoma/leukemia models; however RUNX2 was also shown to play a role in invasive bone, breast, prostate, thyroid and pancreatic cancer. Lately, RUNX2 expression was also associated with EOC tumor progression and poor prognosis. This prompted us to investigate if RUNX2 is induced due to hypomethylation in advanced EOC and whether the RUNX2 gene is functionally implicated in EOC tumorigenesis, including disease progression and response to treatment. Here we show that, similar to RUNX1, the RUNX2 gene is functionally involved in EOC cell proliferation, migration and invasion. However, we also demonstrate that RUNX1 and RUNX2 employ molecular mechanisms in EOC dissemination that are specific for each gene. Snap frozen and formalin-fixed paraffin-embedded tissues of 117 serous EOC tumors were provided by the Banque de tissus et de donne��es of the Re��seau de recherche sur le cancer of the Fonds de recherche du Que��bec – Sante�� at the Hotel-Dieu de Quebec Hospital, Quebec, Canada, which is affiliated with the Canadian Tumor Repository Network. These clinical specimens included 13 borderline, or low-malignant potential tumors, 52 high-grade adenocarcinomas and 52 omental metastases. None of the patients received chemotherapy before surgery. All tumors were histologically classified according to the criteria defined by the World Health Organization. The CT treatment was completed for all patients and the response to treatment was known. Disease progression was evaluated following the guidelines of the Gynecology Cancer Intergroup. Progression free survival was defined as the time from surgery to the first observation of disease progression, recurrence or death. Thirteen normal ovarian samples and 13 normal uterine smooth muscle samples were derived from women subjected to hysterectomy with oophorectomy due to non-ovarian pathologies. TMAs were constructed, as previously described. Briefly, one representative block of each ovarian tumor and normal ovarian tissue was selected for the preparation of the tissue arrays. Three 0.6 mm cores of tumor were taken from each tumor block and placed, 0.4 mm apart, on a recipient paraffin block using a commercial tissue arrayer. The cores were randomly placed on one of two recipient blocks to avoid IHC evaluation biases. Four micron thick sections were cut for the hematoxylin-eosin staining and IHC analyses. IHC was performed, as previously described. Briefly, 4 mm tissue sections were deparaffinized and then heated in an autoclave for 12 min to retrieve the antigenicity before blocking with endogenous peroxidase. We investigated the impact of RUNX2 gene suppression on SKOV3 cell proliferation, cell cycle control, migration, invasion and sensitivity to cisplatin and paclitaxel. The RUNX2 gene knockdown led to a sharp decrease of the number of viable adherent cells, compared to control cells. This observation was further supported by the colony formation assay showing that the numbers of Albaspidin-AA clones formed by cells with stably reduced RUNX2 expression were significantly lower than that of control cells. Taken together, our observations strongly indicate an influence of RUNX2 transcripts on EOC cell proliferation and further on their Tulathromycin B propensity to form colonies. Moreover, RUNX2 suppression significantly inhibited both migration and invasion of SKOV3 cells. As shown in Figure 5A and 5B, the numbers of SKOV3 cells that passed through the filter using shRNA clones 3 and 6 were remarkably less than that in the control clone, which is indicative for a role for RUNX2 in the regulation of invasion and migration in EOC. Similar results were obtained upon RUNX2 knockdown in A2780s cells. Finally, RUNX2 suppression had no significant impact on SKOV3 cell cycle control and cisplatin and paclitaxel sensitivity.

With sumoylation of Ndc10 being functionally relevant. Sumoylation-deficient Ndc10 fails to localize to the mitotic spindle, resulting in defective chromosome segregation. The short defined point centromeres to which Ndc10 binds are unique to the Saccharomycetaceae family of budding yeast, and were recently proposed to have arisen by TWS119 601514-19-6 replacement of a typical epigenetic fungal centromere with an ancestral 2 mm plasmid-derived partitioning system. While rapid evolution may have obscured sequence homology between the 2 mm plasmid and chromosomal segregation proteins, it is tempting to speculate that post-translational modification of segregation proteins with SUMO might be a conserved process, essential for their common function. The yeast 2 mm plasmid is not the only parasitic DNA element to exploit the host cell SUMO pathway for its maintenance. Many viral proteins involved in maintenance of the episomes that encode them are SUMO-modified. Members of the human papillomavirus E2 family of proteins are dependent on sumoylation for their ability to tether viral genomes to host chromosomes to ensure faithful segregation. Host sumoylation has therefore frequently been exploited to ensure maintenance of parasitic genomes in eukaryotic cells, and here we have presented evidence that suggests the yeast 2 mm plasmid may also co-opt this essential cellular process to ensure its efficient segregation during host cell division. Adoptive transfer of ex vivo expanded autologous tumorinfiltrating lymphocytes together followed by one to two cycles of high-dose IL-2 therapy has emerged in multiple Phase II clinical trials to be one of the most powerful therapies for unresectable metastatic melanoma. Durable clinical response rates of up to 50% have been consistently reported using a current protocol consisting of a lymphodepleting preconditioning regimen using cyclophosphamide and fludaribine followed by expanded TIL infusion and IL-2. Our group at MD Anderson Cancer Center has recently completed a study on 31 metastatic patients that have failed multiple first- and second- line therapies using this regimen and reported a 48% clinical response rate. Most responding patients have had progression-free survival times after treatment of.15 LY2109761 months, which is longer than those achieved using other therapies, including targeted therapies with MAPK inhibitors. Although other forms of T-cell therapy have become available, TIL therapy has still remained the superior form of therapy for melanoma because it targets many tumor antigens recognized by a more heterogenous population of T cells rather than a single antigen that can be lost due to the high mutation rates in melanomas. The current method to generate the final TIL product for infusion uses a “rapid expansion protocol” consisting of taking TIL initially expanded from tumor fragments with IL-2 alone for 3�C4 weeks and activating them with anti-CD3 in the presence of a large excess of irradiated PBMC feeder cells. The cells are then expanded for 2 weeks by feeding with culture medium and IL-2. The feeder cells presumably provide a source of Fc receptors for anti-CD3 cross-linking as well as some limited growth factors, anti-oxidants, and co-stimulatory factors for TIL expansion.