The transcriptional co-repressor Runx1t1 is essential for MYCN-driven neuroblastoma tumorigenesis.

MYCN oncogene amplification is frequently observed in aggressive childhood neuroblastoma. Using an unbiased large-scale mutagenesis screen in neuroblastoma-prone transgenic mice, we identify a single germline point mutation in the transcriptional corepressor Runx1t1, which abolishes MYCN-driven tumorigenesis. This loss-of-function mutation disrupts a highly conserved zinc finger domain within Runx1t1. Deletion of one Runx1t1 allele in an independent Runx1t1 knockout mouse model is also sufficient to prevent MYCN-driven neuroblastoma development, and reverse ganglia hyperplasia, a known pre-requisite for tumorigenesis. Silencing RUNX1T1 in human neuroblastoma cells decreases colony formation in vitro, and inhibits tumor growth in vivo. Moreover, RUNX1T1 knockdown inhibits the viability of PAX3-FOXO1 fusion-driven rhabdomyosarcoma and MYC-driven small cell lung cancer cells. Despite the role of Runx1t1 in MYCN-driven tumorigenesis neither gene directly regulates the other. We show RUNX1T1 forms part of a transcriptional LSD1-CoREST3-HDAC repressive complex recruited by HAND2 to enhancer regions to regulate chromatin accessibility and cell-fate pathway genes.

Nutritional considerations in major depressive disorder: current evidence and functional testing for clinical practice.

Depression is a multifaceted condition with diverse underlying causes. Several contributing and inter-related factors such as genetic, nutritional, neurological, physiological, gut-brain-axis, metabolic and psychological stress factors play a role in the pathophysiology of depression. This review aims to highlight the role that nutritional factors play in the aetiology of depression. Secondly, we discuss the biomedical and functional pathology tests which measure these factors, and the current evidence supporting their use. Lastly, we make recommendations on how practitioners can incorporate the latest evidence-based research findings into clinical practice. This review highlights that diet and nutrition greatly affect the pathophysiology of depression. Nutrients influence gene expression, with folate and vitamin B12 playing vital roles in methylation reactions and homocysteine regulation. Nutrients are also involved in the tryptophan/kynurenine pathway and the expression of brain-derived neurotrophic factor (BDNF). Additionally, diet influences the hypothalamic-pituitary-adrenal (HPA) response and the composition and diversity of the gut microbiome, both of which have been implicated in depression. A comprehensive dietary assessment, combined with appropriate evaluation of biochemistry and blood pathology, may help uncover contributing factors to depressive symptoms. By employing such an approach, a more targeted and personalised treatment strategy can be devised, ultimately leading to improved patient outcomes.

Collaborative Active Learning Activities Promote Deep Learning in a Chemistry-Biochemistry Course.

Currently in higher education, there is a move towards providing more student-centred learning experiences, where students are actively involved in the learning process. To promote learner engagement and communication between peers, many educators utilise collaborative active learning activities. This study aimed to demonstrate that an active learning curriculum developed for a Chemistry-Biochemistry unit, allowed students to gain a deep understanding of the content, while developing key academic skills. In each face-to-face session of the Chemistry-Biochemistry unit, students participated in collaborative active learning activities including Participation+ and a variety of Padlet activities. The students were also challenged to develop their written communication skills, by taking part in a formative In-Class Writing Task. Survey results indicated that the active learning curriculum provided an engaging, interactive environment that was conducive to the students developing an understanding of the course's underlying concepts and developing key academic skills. The students communicated their deep understanding of the content verbally during active learning activities and in writing during the In-Class Writing Task, written assignment and final exam. Students who consistently communicated deep knowledge of the content during the In-Class Writing Task achieved high marks on the summative written assignment, final exam and unit total. This study clearly demonstrates that the active learning curriculum employed in the Chemistry-Biochemistry unit provided a collaborative and engaging learning environment, where many students developed a deep understanding of the content and acquired the skills to communicate their knowledge both orally and through written communication.

Glutathione biosynthesis is upregulated at the initiation of MYCN-driven neuroblastoma tumorigenesis.

The MYCN gene is amplified and overexpressed in a large proportion of high stage neuroblastoma patients and has been identified as a key driver of tumorigenesis. However, the mechanism by which MYCN promotes tumor initiation is poorly understood. Here we conducted metabolic profiling of pre-malignant sympathetic ganglia and tumors derived from the TH-MYCN mouse model of neuroblastoma, compared to non-malignant ganglia from wildtype littermates. We found that metabolites involved in the biosynthesis of glutathione, the most abundant cellular antioxidant, were the most significantly upregulated metabolic pathway at tumor initiation, and progressively increased to meet the demands of tumorigenesis. A corresponding increase in the expression of genes involved in ribosomal biogenesis suggested that MYCN-driven transactivation of the protein biosynthetic machinery generated the necessary substrates to drive glutathione biosynthesis. Pre-malignant sympathetic ganglia from TH-MYCN mice had higher antioxidant capacity and required glutathione upregulation for cell survival, when compared to wildtype ganglia. Moreover, in vivo administration of inhibitors of glutathione biosynthesis significantly delayed tumorigenesis when administered prophylactically and potentiated the anticancer activity of cytotoxic chemotherapy against established tumors. Together these results identify enhanced glutathione biosynthesis as a selective metabolic adaptation required for initiation of MYCN-driven neuroblastoma, and suggest that glutathione-targeted agents may be used as a potential preventative strategy, or as an adjuvant to existing chemotherapies in established disease.

Management of knee osteoarthritis by combined stromal vascular fraction cell therapy, platelet-rich plasma, and musculoskeletal exercises: a case series.

INTRODUCTION: Knee osteoarthritis is associated with persistent joint pain, stiffness, joint deformities, ligament damage, and surrounding muscle atrophy. The complexity of the disease makes treatment difficult. There are no therapeutic drugs available to halt the disease progression, leaving patients dependent on pain medication, anti-inflammatory drugs, or invasive joint replacement surgery. CASE PRESENTATIONS: Four patients with a history of unresolved symptomatic knee osteoarthritis were investigated for the therapeutic outcome of combining an exercise rehabilitation program with intra-articular injections of autologous StroMed (ie, stromal vascular fraction cells concentrated by ultrasonic cavitation from lipoaspirate) and platelet-rich plasma (PRP). The Knee Injury and Osteoarthritis Outcome Score questionnaire (KOOS) was administered along with physical function tests over a 12-month period. The first patient achieved a maximum therapeutic outcome of 100 in all five KOOS subscales (left knee), and 100 for four subscales (right knee). The second patient scored 100 in all five KOOS subscales (left knee), and greater than 84 in all subscales (right knee). Treatment of the third patient resulted in improved outcomes in both knees of >93 for four KOOS subscales, and 60 for the Function in Sport and Recreation subscale. The fourth patient improved to 100 in all five KOOS subscales. In all patients, the physical function "Get-up and Go" test and "Stair Climbing Test" returned to normal (a value of zero). CONCLUSION: This case series indicates that improved outcomes may be obtained when autologous stromal vascular fraction (StroMed) cell therapy is combined with traditional exercise practices and PRP for osteoarthritis. Of the seven joints treated: all patients' scores of pain improved to >96; and quality of life scores to >93. Functional performance measures of mobility returned to normal. This simple treatment appears to be extremely effective for osteoarthritis disorders that have no drug treatment to halt disease progression.

The retinoid signalling molecule, TRIM16, is repressed during squamous cell carcinoma skin carcinogenesis in vivo and reduces skin cancer cell migration in vitro.

Retinoid therapy is used for chemo-prevention in immuno-suppressed patients at high risk of developing skin cancer. The retinoid signalling molecule, tripartite motif protein 16 (TRIM16), is a regulator of keratinocyte differentiation and a tumour suppressor in retinoid-sensitive neuroblastoma. We sought to determine the role of TRIM16 in skin squamous cell carcinoma (SCC) pathogenesis. We have shown that TRIM16 expression was markedly reduced during the histological progression from normal skin to actinic keratosis and SCC. SCC cell lines exhibited lower cytoplasmic and nuclear TRIM16 expression compared with primary human keratinocyte (PHK) cells due to reduced TRIM16 protein stability. Overexpressed TRIM16 translocated to the nucleus, inducing growth arrest and cell differentiation. In SCC cells, TRIM16 bound to and down regulated nuclear E2F1, this is required for cell replication. Retinoid treatment increased nuclear TRIM16 expression in retinoid-sensitive PHK cells, but not in retinoid-resistant SCC cells. Overexpression of TRIM16 reduced SCC cell migration, which required the C-terminal RET finger protein (RFP)-like domain of TRIM16. The mesenchymal intermediate filament protein, vimentin, was directly bound and down-regulated by TRIM16 and was required for TRIM16-reduced cell migration. Taken together, our data suggest that loss of TRIM16 expression plays an important role in the development of cutaneous SCC and is a determinant of retinoid sensitivity.

SIRT1 promotes N-Myc oncogenesis through a positive feedback loop involving the effects of MKP3 and ERK on N-Myc protein stability.

The N-Myc oncoprotein is a critical factor in neuroblastoma tumorigenesis which requires additional mechanisms converting a low-level to a high-level N-Myc expression. N-Myc protein is stabilized when phosphorylated at Serine 62 by phosphorylated ERK protein. Here we describe a novel positive feedback loop whereby N-Myc directly induced the transcription of the class III histone deacetylase SIRT1, which in turn increased N-Myc protein stability. SIRT1 binds to Myc Box I domain of N-Myc protein to form a novel transcriptional repressor complex at gene promoter of mitogen-activated protein kinase phosphatase 3 (MKP3), leading to transcriptional repression of MKP3, ERK protein phosphorylation, N-Myc protein phosphorylation at Serine 62, and N-Myc protein stabilization. Importantly, SIRT1 was up-regulated, MKP3 down-regulated, in pre-cancerous cells, and preventative treatment with the SIRT1 inhibitor Cambinol reduced tumorigenesis in TH-MYCN transgenic mice. Our data demonstrate the important roles of SIRT1 in N-Myc oncogenesis and SIRT1 inhibitors in the prevention and therapy of N-Myc-induced neuroblastoma.

Serial transcriptome analysis and cross-species integration identifies centromere-associated protein E as a novel neuroblastoma target.

Cancer genomic studies that rely on analysis of biopsies from primary tumors may not fully identify the molecular events associated with tumor progression. We hypothesized that characterizing the transcriptome during tumor progression in the TH-MYCN transgenic model would identify oncogenic drivers that would be targetable therapeutically. We quantified expression of 32,381 murine genes in nine hyperplastic ganglia harvested at three time points and four tumor cohorts of progressively larger size in mice homozygous for the TH-MYCN transgene. We found 93 genes that showed a linearly increasing or decreasing pattern of expression from the preneoplastic ganglia to end stage tumors. Cross-species integration identified 24 genes that were highly expressed in human MYCN-amplified neuroblastomas. The genes prioritized were not exclusively driven by increasing Myc transactivation or proliferative rate. We prioritized three targets [centromere-associated protein E (Cenpe), Gpr49, and inosine monophosphate dehydrogenase type II] with previously determined roles in cancer. Using siRNA knockdown in human neuroblastoma cell lines, we further prioritized CENPE due to inhibition of cellular proliferation. Targeting CENPE with the small molecular inhibitor GSK923295 showed inhibition of in vitro proliferation of 19 neuroblastoma cell lines (median IC(50), 41 nmol/L; range, 27-266 nmol/L) and delayed tumor growth in three xenograft models (P values ranged from P < 0.0001 to P = 0.018). We provide preclinical validation that serial transcriptome analysis of a transgenic mouse model followed by cross-species integration is a useful method to identify therapeutic targets and identify CENPE as a novel therapeutic candidate in neuroblastoma.

The estrogen-responsive B box protein (EBBP) restores retinoid sensitivity in retinoid-resistant cancer cells via effects on histone acetylation.

Retinoids have significant clinical activity in several human cancers, yet the factors determining retinoid sensitivity in cancer cells are still unclear. Retinoid-induced expression of retinoic acid receptor (RAR) beta(2) is a necessary component of the retinoid anticancer signal in cancer cells. We have previously identified the Estrogen-responsive B Box Protein (EBBP), a member of the Tripartite Motif (TRIM) protein family, as a novel RARbeta2 transcriptional regulator in the retinoid signal. Here we examined the mechanism of the EBBP effect on the retinoid anticancer signal. We assessed retinoid-responsive RARbeta2 transcription in retinoid-resistant breast and lung cancer cells in the presence of chromatin modifying agents. A histone deacetylase (HDAC) inhibitor alone, or in combination with retinoid, was more effective than a demethylating agent in restoring RARbeta2 transcription in resistant cells. Overexpression of EBBP alone markedly increased histone acetylation. The effect of EBBP on retinoid-responsive transcription appeared to be limited to genes with the retinoic acid response element (betaRARE) regulatory sequence, such as CYP26A1. EBBP inhibited cell growth by effects on cyclin D1 and Phospho-Rb, and, reduced cell viability in retinoid-resistant cancer cells. The viability of non-cancer cells was unaffected by EBBP overexpression. Taken together our data suggests that EBBP acts to de-repress transcription of RARbeta2 and CYP26A1, by modifying histone acetylation in retinoid-resistant cancer cells, and, is an important target for drug discovery in retinoid-resistant cancers.

ODC1 is a critical determinant of MYCN oncogenesis and a therapeutic target in neuroblastoma.

Neuroblastoma is a frequently lethal childhood tumor in which MYC gene deregulation, commonly as MYCN amplification, portends poor outcome. Identifying the requisite biopathways downstream of MYC may provide therapeutic opportunities. We used transcriptome analyses to show that MYCN-amplified neuroblastomas have coordinately deregulated myriad polyamine enzymes (including ODC1, SRM, SMS, AMD1, OAZ2, and SMOX) to enhance polyamine biosynthesis. High-risk tumors without MYCN amplification also overexpress ODC1, the rate-limiting enzyme in polyamine biosynthesis, when compared with lower-risk tumors, suggesting that this pathway may be pivotal. Indeed, elevated ODC1 (independent of MYCN amplification) was associated with reduced survival in a large independent neuroblastoma cohort. As polyamines are essential for cell survival and linked to cancer progression, we studied polyamine antagonism to test for metabolic dependence on this pathway in neuroblastoma. The Odc inhibitor alpha-difluoromethylornithine (DFMO) inhibited neuroblast proliferation in vitro and suppressed oncogenesis in vivo. DFMO treatment of neuroblastoma-prone genetically engineered mice (TH-MYCN) extended tumor latency and survival in homozygous mice and prevented oncogenesis in hemizygous mice. In the latter, transient Odc ablation permanently prevented tumor onset consistent with a time-limited window for embryonal tumor initiation. Importantly, we show that DFMO augments antitumor efficacy of conventional cytotoxics in vivo. This work implicates polyamine biosynthesis as an arbiter of MYCN oncogenesis and shows initial efficacy for polyamine depletion strategies in neuroblastoma, a strategy that may have utility for this and other MYC-driven embryonal tumors.

Activation of tissue transglutaminase transcription by histone deacetylase inhibition as a therapeutic approach for Myc oncogenesis.

Histone deacetylase (HDAC) inhibitors reactivate tumor suppressor gene transcription; induce cancer cell differentiation, growth arrest, and programmed cell death; and are among the most promising new classes of anticancer drugs. Myc oncoproteins can block cell differentiation and promote cell proliferation and malignant transformation, in some cases by modulating target gene transcription. Here, we show that tissue transglutaminase (TG2) was commonly reactivated by HDAC inhibitors in neuroblastoma and breast cancer cells but not normal cells and contributed to HDAC inhibitor-induced growth arrest. TG2 was the gene most significantly repressed by N-Myc in neuroblastoma cells in a cDNA microarray analysis and was commonly repressed by N-Myc in neuroblastoma cells and c-Myc in breast cancer cells. Repression of TG2 expression by N-Myc in neuroblastoma cells was necessary for the inhibitory effect of N-Myc on neuroblastoma cell differentiation. Dual step cross-linking chromatin immunoprecipitation and protein coimmunoprecipitation assays showed that N-Myc acted as a transrepressor by recruiting the HDAC1 protein to an Sp1-binding site in the TG2 core promoter in a manner distinct from it's action as a transactivator at E-Box binding sites. HDAC inhibitor treatment blocked the N-Myc-mediated HDAC1 recruitment and TG2 repression in vitro. In neuroblastoma-bearing N-Myc transgenic mice, HDAC inhibitor treatment induced TG2 expression and demonstrated marked antitumor activity in vivo. Taken together, our data indicate the critical roles of HDAC1 and TG2 in Myc-induced oncogenesis and have significant implications for the use of HDAC inhibitor therapy in Myc-driven oncogenesis.

Role of melanotransferrin in iron metabolism: studies using targeted gene disruption in vivo.

Melanotransferrin (MTf) or tumor antigen p97 is a transferrin homolog that binds one iron (Fe) atom and has been suggested to play roles in a variety of processes, including Fe metabolism, eosinophil differentiation, and plasminogen activation. Considering the vital role of Fe in many metabolic pathways, such as DNA and heme synthesis, it is important to understand the function of MTf. To define this, a MTf knockout (MTf-/-) mouse was generated through targeted disruption of the MTf gene. The MTf-/- mice were viable and fertile and developed normally, with no morphologic or histologic abnormalities. Assessment of Fe indices, tissue Fe levels, hematology, and serum chemistry parameters demonstrated no differences between MTf-/- and wild-type (MTf+/+) mice, suggesting MTf was not essential for Fe metabolism.

Giardia intestinalis: molecular characterization of UDP-N-acetylglucosamine pyrophosphorylase.

The flagellated protozoan Giardia intestinalis is one of the most prevalent human-infective parasites with a worldwide distribution. This parasite must encyst to complete the life cycle and N-acetylgalactosamine is produced from endogenous glucose for cyst wall synthesis during the transformation. UDP-N-acetylglucosamine pyrophosphorylase in G. intestinalis (GiUAP, EC 2.7.7.23) is the fourth enzyme in the inducible pathway of N-acetylgalactosamine biosynthesis, catalysing the conversion of N-acetylglucosamine-1-P to UDP-N-acetylglucosamine. In this study the gene GiUAP was cloned and sequenced from the Portland 1 strain using PCR techniques. It has an ORF of approximately 1.3 kb and contains no introns. BLAST and ClustalW analysis of the deduced amino acid sequence revealed significant similarities to other eukaryotic UAPs with putative active sites identified. Southern hybridization showed that GiUAP exists as a single-copy gene and it was shown to have two transcripts by RT-PCR and Northern hybridization. RLM-RACE identified both 5' and 3' untranslated regions and suggested the transcripts exist as a 5'-capped mRNA, with the use of two tandem polyadenylation sites to generate two unusually long giardial 3' untranslated regions of approximately 522 bp and approximately 3 kb. Moreover, a recombinant protein (rGiUAP) was expressed in E. coli and subjected to physical characterizations. Surprisingly the results obtained in this study were significantly different from those reported for the GiUAP in MR4 strain, suggesting this gene is under different transcription control in different strains of G. intestinalis. This report describes the molecular characterization of GiUAP and provides an opportunity to explore the control of gene expression during encystation of the parasite.

The soluble form of the membrane-bound transferrin homologue, melanotransferrin, inefficiently donates iron to cells via nonspecific internalization and degradation of the protein.

Melanotransferrin (MTf) is a membrane-bound transferrin (Tf) homologue found particularly in melanoma cells. Apart from membrane-bound MTf, a soluble form of the molecule (sMTf) has been identified in vitro[Food, M.R., Rothenberger, S., Gabathuler, R., Haidl, I.D., Reid, G. & Jefferies, W.A. (1994) J. Biol. Chem.269, 3034-3040] and in vivo in Alzheimer's disease. However, nothing is known about the function of sMTf or its role in Fe uptake. In this study, sMTf labelled with 59Fe and 125I was used to examine its ability to donate 59Fe to SK-Mel-28 melanoma cells and other cell types. sMTf donated 59Fe to cells at 14% of the rate of Tf. Analysis of sMTf binding showed that unlike Tf, sMTf did not bind to a saturable Tf-binding site. Studies with Chinese hamster ovary cells with and without specific Tf receptors showed that unlike Tf, sMTf did not donate its 59Fe via these pathways. This was confirmed by experiments using lysosomotropic agents that markedly reduced 59Fe uptake from Tf, but had far less effect on 59Fe uptake from sMTf. In addition, an excess of 56Fe-labelled Tf or sMTf had no effect on 125I-labelled sMTf uptake, suggesting a nonspecific interaction of sMTf with cells. Protein-free 125I determinations demonstrated that in contrast with Tf, sMTf was markedly degraded. We suggest that unlike the binding of Tf to specific receptors, sMTf was donating Fe to cells via an inefficient mechanism involving nonspecific internalization and subsequent degradation.