ASCT2 regulates glutamine uptake and cell growth in endometrial carcinoma.

Glutamine commonly becomes a conditionally essential amino acid in cancer. Glutamine is supplied to the cell by transporters such as ASCT2 (SLC1A5), which is frequently upregulated in multiple cancers. Here we investigated the expression of ASCT2 in endometrial carcinoma, and evaluated the contribution of ASCT2 to glutamine uptake and endometrial cancer cell growth. Analysis of human gene expression data showed that ASCT2 was significantly upregulated in both endometrioid and serous subtypes of endometrial carcinoma, compared to normal, age-matched endometrium. Furthermore, immunohistochemical staining of primary human endometrioid adenocarcinomas showed that tumours stain positive for ASCT2 in either a uniform or mosaic expression pattern, while normal adjacent glands appeared predominantly negative for ASCT2 staining. Chemical inhibition of glutamine transport by benzylserine or GPNA led to a significant decrease in endometrial cancer cell growth and spheroid cross-sectional area. ASCT2 knockdown recapitulated the decrease of cell growth and spheroid cross-sectional area in HEC1A cells, suggesting a reliance on ASCT2-mediated glutamine uptake. ASCT2 knockdown in Ishikawa cells led to lower glutamine uptake and cell growth, but did not affect spheroid area. Ishikawa cells express higher levels of the glutamine transporter SNAT1 compared to HEC1A cells, suggesting these cells may rely on both ASCT2 and SNAT1 for glutamine uptake. Since SNAT1 is also significantly upregulated in the endometrioid and serous subtypes, these data indicate that ASCT2 and SNAT1 could be used as markers of malignancy, and/or potential therapeutic targets in patients with endometrial carcinoma.

CTCF genetic alterations in endometrial carcinoma are pro-tumorigenic.

CTCF is a haploinsufficient tumour suppressor gene with diverse normal functions in genome structure and gene regulation. However the mechanism by which CTCF haploinsufficiency contributes to cancer development is not well understood. CTCF is frequently mutated in endometrial cancer. Here we show that most CTCF mutations effectively result in CTCF haploinsufficiency through nonsense-mediated decay of mutant transcripts, or loss-of-function missense mutation. Conversely, we identified a recurrent CTCF mutation K365T, which alters a DNA binding residue, and acts as a gain-of-function mutation enhancing cell survival. CTCF genetic deletion occurs predominantly in poor prognosis serous subtype tumours, and this genetic deletion is associated with poor overall survival. In addition, we have shown that CTCF haploinsufficiency also occurs in poor prognosis endometrial clear cell carcinomas and has some association with endometrial cancer relapse and metastasis. Using shRNA targeting CTCF to recapitulate CTCF haploinsufficiency, we have identified a novel role for CTCF in the regulation of cellular polarity of endometrial glandular epithelium. Overall, we have identified two novel pro-tumorigenic roles (promoting cell survival and altering cell polarity) for genetic alterations of CTCF in endometrial cancer.

Experimental approaches to studying the nature and impact of splicing variation in zebrafish.

From a fixed number of genes carried in all cells, organisms create considerable diversity in cellular phenotype through differential regulation of gene expression. One prevalent source of transcriptome diversity is alternative pre-mRNA splicing, which is manifested in many different forms. Zebrafish models of splicing dysfunction due to mutated spliceosome components provide opportunity to link biochemical analyses of spliceosome structure and function with whole organism phenotypic outcomes. Drawing from experience with two zebrafish mutants: cephalophonus (a prpf8 mutant, isolated for defects in granulopoiesis) and caliban (a rnpc3 mutant, isolated for defects in digestive organ development), we describe the use of glycerol gradient sedimentation and native gel electrophoresis to resolve components of aberrant splicing complexes. We also describe how RNAseq can be employed to examine relatively rare alternative splicing events including intron retention. Such experimental approaches in zebrafish can promote understanding of how splicing variation and dysfunction contribute to phenotypic diversity and disease pathogenesis.

ASCT2/SLC1A5 controls glutamine uptake and tumour growth in triple-negative basal-like breast cancer.

Alanine, serine, cysteine-preferring transporter 2 (ASCT2; SLC1A5) mediates uptake of glutamine, a conditionally essential amino acid in rapidly proliferating tumour cells. Uptake of glutamine and subsequent glutaminolysis is critical for activation of the mTORC1 nutrient-sensing pathway, which regulates cell growth and protein translation in cancer cells. This is of particular interest in breast cancer, as glutamine dependence is increased in high-risk breast cancer subtypes. Pharmacological inhibitors of ASCT2-mediated transport significantly reduced glutamine uptake in human breast cancer cell lines, leading to the suppression of mTORC1 signalling, cell growth and cell cycle progression. Notably, these effects were subtype-dependent, with ASCT2 transport critical only for triple-negative (TN) basal-like breast cancer cell growth compared with minimal effects in luminal breast cancer cells. Both stable and inducible shRNA-mediated ASCT2 knockdown confirmed that inhibiting ASCT2 function was sufficient to prevent cellular proliferation and induce rapid cell death in TN basal-like breast cancer cells, but not in luminal cells. Using a bioluminescent orthotopic xenograft mouse model, ASCT2 expression was then shown to be necessary for both successful engraftment and growth of HCC1806 TN breast cancer cells in vivo. Lower tumoral expression of ASCT2 conferred a significant survival advantage in xenografted mice. These responses remained intact in primary breast cancers, where gene expression analysis showed high expression of ASCT2 and glutamine metabolism-related genes, including GLUL and GLS, in a cohort of 90 TN breast cancer patients, as well as correlations with the transcriptional regulators, MYC and ATF4. This study provides preclinical evidence for the feasibility of novel therapies exploiting ASCT2 transporter activity in breast cancer, particularly in the high-risk basal-like subgroup of TN breast cancer where there is not only high expression of ASCT2, but also a marked reliance on its activity for sustained cellular proliferation.

Clinical potential of gene therapy: towards meeting the demand.

Since the discovery that new genetic material could be transferred into human cells resulting in induced expression of genes and proteins, clinicians and scientists have been working to harness the technology for clinical outcomes. This article provides a summary of the current status of developments within the broad discipline of clinical gene therapy. In pursuing the treatment of diverse clinical conditions, a wide variety of therapeutics, each tailor-made, may be required. Gene therapy offers the possibility of accurately and specifically targeting particular genetic abnormalities through gene correction, addition or replacement. It represents a compelling idea that adds a new dimension to our portfolio of credible therapeutic choices.

Circulating tumour cells and circulating free nucleic acid as prognostic and predictive biomarkers in colorectal cancer.

The detection of circulating tumour cells or circulating free tumour nucleic acids can potentially guide treatment and inform prognosis in colorectal cancer using minimally invasive "liquid biopsies". Current literature supports the notion that high circulating tumour cell counts or presence of tumour nucleic acid correlate with inferior clinical outcomes for patients, but they are not yet part of routine clinical care. Future research evolves around the examination of the molecular phenotype of circulating tumour cells. The key unanswered areas include differentiating between circulating tumour cell presence and their proliferative capacity and dormancy, identifying tumour heterogeneity and understanding the epithelial-mesenchymal transition.

Performance evaluation of the Abbott CELL-DYN Emerald for use as a bench-top analyzer in a research setting.

INTRODUCTION: The CELL-DYN Emerald is a compact bench-top hematology analyzer that can be used for a three-part white cell differential analysis. To determine its utility for analysis of human and mouse samples, we evaluated this machine against the larger CELL-DYN Sapphire and Sysmex XT2000iV hematology analyzers. METHODS: 120 human (normal and abnormal) and 30 mouse (normal and abnormal) samples were analyzed on both the CELL-DYN Emerald and CELL-DYN Sapphire or Sysmex XT2000iV analyzers. For mouse samples, the CELL-DYN Emerald analyzer required manual recalibration based on the histogram populations. RESULTS: Analysis of the CELL-DYN Emerald showed excellent precision, within accepted ranges (white cell count CV% = 2.09%; hemoglobin CV% = 1.68%; platelets CV% = 4.13%). Linearity was excellent (R² ≥ 0.99), carryover was minimal (<1%), and overall interinstrument agreement was acceptable for both human and mouse samples. Comparison between the CELL-DYN Emerald and Sapphire analyzers for human samples or Sysmex XT2000iV analyzer for mouse samples showed excellent correlation for all parameters. CONCLUSION: The CELL-DYN Emerald was generally comparable to the larger reference analyzer for both human and mouse samples. It would be suitable for use in satellite research laboratories or as a backup system in larger laboratories.

Loss of solute carriers in T cell-mediated rejection in mouse and human kidneys: an active epithelial injury-repair response.

T cell-mediated rejection of kidney allografts causes epithelial deterioration, manifested by tubulitis, but the mechanism remains unclear. We hypothesized that interstitial inflammation triggers a stereotyped epithelial response similar to that triggered by other types of injury such as ischemia-reperfusion. We identified solute carrier transcripts with decreased expression in mouse allografts, and compared their behavior in T cell-mediated rejection to native kidneys with ischemic acute tubular necrosis (ATN). Average loss of solute carrier expression was similar in ATN (77%) and T cell-mediated rejection (75%) with high correlation of individual transcripts. Immunostaining of SLC6A19 confirmed loss of proteins. Analysis of human kidney transplant biopsies confirmed that T cell-mediated rejection and ATN showed similar loss of solute carrier mRNAs. The loss of solute carrier expression was weakly correlated with interstitial inflammation, but kidneys with ATN showed decreased solute carriers despite minimal inflammation. Loss of renal function correlated better with decreased solute carrier expression than with histologic lesions (r = 0.396, p < 0.001). Thus the loss of epithelial transcripts in rejection is not a unique consequence of T cell-mediated rejection but an active injury-repair response of epithelium, triggered by rejection but also by other injury mechanisms.

A prospective randomized, controlled trial of intravenous versus oral iron for moderate iron deficiency anaemia of pregnancy.

BACKGROUND: Iron deficiency anaemia is the most common deficiency disorder in the world, affecting more than one billion people, with pregnant women at particular risk. OBJECTIVES AND DESIGN: We conducted a single site, prospective, nonblinded randomized-controlled trial to compare the efficacy, safety, tolerability and compliance of standard oral daily iron versus intravenous iron. SUBJECTS: We prospectively screened 2654 pregnant women between March 2007 and January 2009 with a full blood count and iron studies, of which 461 (18%) had moderate IDA. Two hundred women matched for haemoglobin concentration and serum ferritin level were recruited. INTERVENTIONS: Patients were randomized to daily oral ferrous sulphate 250 mg (elemental iron 80 mg) with or without a single intravenous iron polymaltose infusion. RESULTS: Prior to delivery, the intravenous plus oral iron arm was superior to the oral iron only arm as measured by the increase in haemoglobin level (mean of 19.5 g/L vs. 12 g/L; P < 0.001); the increase in mean serum ferritin level (222 microg/L vs. 18 ug/L; P < 0.001); and the percentage of mothers with ferritin levels below 30 microg/L (4.5% vs. 79%; P < 0.001). A single dose of intravenous iron polymaltose was well tolerated without significant side effects. CONCLUSIONS: Our data indicate that intravenous iron polymaltose is safe and leads to improved efficacy and iron stores compared to oral iron alone in pregnancy-related IDA.

Distribution of human endogenous retrovirus type W receptor in normal human villous placenta.

BACKGROUND: The fusion of trophoblast cells into the villous syncytiotrophoblast is crucial for appropriate placental function and fetal development. Fusion occurs following the interaction of syncytin-1, an envelope protein of the endogenous retrovirus HERV-W, and the RD114/mammalian type D retrovirus receptor (RDR/ASCT2) on adjacent cell membranes. This process must be tightly regulated in order to maintain the proliferative pool of cytotrophoblast cells as well as the function of the syncytia. AIM: We sought to investigate whether syncytial fusion of placental cytotrophoblast cells may be regulated via modulation of RDR/ASCT2 expression. METHODS: Expression of RDR/ASCT2 in term and first trimester villous placenta was assessed along with a number of molecular markers using immunofluorescent staining. In a complementary approach, Western blotting was used to investigate RDR/ASCT2 expression in a panel of choriocarcinoma cell lines before and after stimulation of fusion. RESULTS: Villous placental RDR/ASCT2 expression was found to be restricted to the cytotrophoblast compartment, being largely absent in the syncytiotrophoblast. Local variations in RDR/ASCT2 expression were not associated with the proliferative status of cytotrophoblast cells. RDR/ASCT2 expression was also shown to be down-regulated in BeWo choriocarcinoma cells after stimulation of syncytial fusion. CONCLUSION: This first report of the localisation and distribution of RDR/ASCT2 in human placental villi suggests that the fusion of placental trophoblast cells is not regulated by local or temporal variations of RDR/ASCT2 expression in villous cytotrophoblast cells.

Mobilization of bone marrow-derived progenitors.

Bone marrow (BM) is a source of various stem and progenitor cells in the adult, and it is able to regenerate a variety of tissues following transplantation. In the 1970s the first BM stem cells identified were hematopoietic stem cells (HSCs). HSCs have the potential to differentiate into all myeloid (including erythroid) and lymphoid cell lineages in vitro and reconstitute the entire hematopoietic and immune systems following transplantation in vivo. More recently, nonhematopoietic stem and progenitor cells have been identified that can differentiate into other cell types such as endothelial progenitor cells (EPCs), contributing to the neovascularization of tumors as well as ischemic tissues, and mesenchymal stem cells (MSCs), which are able to differentiate into many cells of ectodermal, endodermal, and mesodermal origins in vitro as well as in vivo. Following adequate stimulation, stem and progenitor cells can be forced out of the BM to circulate into the peripheral blood, a phenomenon called "mobilization." This chapter reviews the molecular mechanisms behind mobilization and how these have led to the various strategies employed to mobilize BM-derived stem and progenitor cells in experimental and clinical settings. Mobilization of HSCs will be reviewed first, as it has been best-explored--being used extensively in clinics to transplant large numbers of HSCs to rescue cancer patients requiring hematopoietic reconstitution--and provides a paradigm that can be generalized to the mobilization of other types of BM-derived stem and progenitor cells in order to repair other tissues.

Cell and gene therapy in Australia.

The expansion of human cells to produce cell therapeutic products for the treatment of disease is, with few exceptions, an experimental therapy. Because cell therapies involve a biological product, often with some genetic or other modification, they require extensive pre-clinical research and development. Cell therapy production processes and premises require licensing by the Therapeutic Goods Administration. In this review, timed to coincide with the international meetings of the ISCT and ISSCR in Australia, we describe some promising cell therapies currently under development.

Neutral amino acid transport in epithelial cells and its malfunction in Hartnup disorder.

Hartnup disorder is an autosomal recessive abnormality of renal and gastrointestinal neutral amino acid transport. A corresponding transport activity has been characterized in kidney and intestinal cells and named system B(0). The failure to resorb amino acids in this disorder is thought to be compensated by a protein-rich diet. However, in combination with a poor diet and other factors, more severe symptoms can develop in Hartnup patients, including a photosensitive pellagra-like skin rash, cerebellar ataxia and other neurological symptoms. Homozygosity mapping in a Japanese family and linkage analysis on six Australian pedigrees placed the Hartnup disorder gene at a locus on chromosome 5p15. This fine mapping facilitated a candidate gene approach within the interval, which resulted in the cloning and characterization of a novel member of the sodium-dependent neurotransmitter transporter family (B(0)AT1, SLC6A19) from mouse and human kidney, which shows all properties of system B(0). Flux experiments and electrophysiological recording showed that the transporter is Na(+) dependent and Cl(-) independent, electrogenic and actively transports most neutral amino acids. In situ hybridization showed strong expression in intestinal villi and in the proximal tubule of the kidney. Expression of B(0)AT1 was restricted to kidney, intestine and skin. A total of ten mutations have been identified in SLC6A19 that co-segregate with disease in the predicted recessive manner, with the majority of affected individuals being compound heterozygotes. These mutations lead to altered neutral amino acid transport function compared to the wild-type allele in vitro. One of the mutations occurs in members of the original Hartnup family described in 1956, thereby defining SLC6A19 as the 'Hartnup'-gene.

Hartnup disorder: polymorphisms identified in the neutral amino acid transporter SLC1A5.

Hartnup disorder is an inborn error of renal and gastrointestinal neutral amino acid transport. The cloning and functional characterization of the 'system B0' neutral amino acid transporter SLC1A5 led to it being proposed as a candidate gene for Hartnup disorder. Linkage analysis performed at 19q13.3, the chromosomal position of SLC1A5, was suggestive of an association with the Hartnup phenotype in some families. However, SLC1A5 was not linked to the Hartnup phenotype in other families. Linkage analysis also excluded an alternative candidate region at 11q13 implicated by a putative mouse model for Hartnup disorder. Sequencing of the coding region of SLC1A5 in Hartnup patients revealed two coding region polymorphisms. These mutations did not alter the predicted amino acid sequence of SLC1A5 and were considered unlikely to play a role in Hartnup disorder. There were no mutations in splice sites flanking each exon. Quantitative RT-PCR of SLC1A5 messenger RNA in affected and unaffected subjects did not support systemic differences in expression as an explanation for Hartnup disorder. In the six unrelated Hartnup pedigrees studied, examination of linkage at 19q13.3, polymorphisms in the coding sequence and quantitation of expression of SLC1A5 did not suffice to explain the defect in neutral amino acid transport.