Jeremy Turner

Leukemic blasts program bone marrow adipocytes to generate a protumoral microenvironment

Despite currently available therapies, most patients diagnosed with acute myeloid leukemia (AML) die of their disease. Tumor-host interactions are critical for the survival and proliferation of cancer cells; accordingly, we hypothesize that specific targeting of the tumor microenvironment may constitute an alternative or additional strategy to conventional tumor-directed chemotherapy. Because adipocytes have been shown to promote breast and prostate cancer proliferation, and because the bone marrow adipose tissue accounts for up to 70% of bone marrow volume in adult humans, we examined the adipocyte-leukemia cell interactions to determine if they are essential for the growth and survival of AML. Using in vivo and in vitro models of AML, we show that bone marrow adipocytes from the tumor microenvironment support the survival and proliferation of malignant cells from patients with AML. We show that AML blasts alter metabolic processes in adipocytes to induce phosphorylation of hormone-sensitive lipase and consequently activate lipolysis, which then enables the transfer of fatty acids from adipocytes to AML blasts. In addition, we report that fatty acid binding protein-4 (FABP4) messenger RNA is upregulated in adipocytes and AML when in coculture. FABP4 inhibition using FABP4 short hairpin RNA knockdown or a small molecule inhibitor prevents AML proliferation on adipocytes. Moreover, knockdown of FABP4 increases survival in Hoxa9/Meis1-driven AML model. Finally, knockdown of carnitine palmitoyltransferase IA in an AML patient-derived xenograft model improves survival. Here, we report the first description of AML programming bone marrow adipocytes to generate a protumoral microenvironment.

Effect of tissue inhibitor of metalloproteinases 3 on DLK1 shedding in cultured human pre-adipocytes and implications for adipose tissue remodelling

BACKGROUND Metabolically unhealthy obesity is associated with adipose tissue inflammation and increased risk of type 2 diabetes. Dysfunctional adipose tissue remodelling has been implicated in development of metabolically unhealthy obesity, but the pathogenesis remains poorly characterised. We hypothesised that in health, tissue inhibitor of metalloproteinases 3 (TIMP3) modulates adipose tissue remodelling by regulating extracellular matrix turnover and shedding of the adipogenic regulator DLK1, but that in adipose tissue inflammation it might drive development of metabolically unhealthy obesity. METHODS Primary pre-adipocyte and in-vitro-differentiated adipocyte cultures were established from abdominal subcutaneous adipose tissue donated by healthy women undergoing breast reconstruction. Cells were seeded onto collagen I, and subsequently treated with differentiation medium or tumour necrosis factor (TNF) alpha (50 ng/mL). Adenoviral transduction allowed TIMP3 overexpression. Media and lysates were collected for quantitative RT-PCR, and immunoblot and hydroxyproline release assays. Statistical analysis was performed with t testing or ANOVA. FINDINGS Induction of differentiation in human pre-adipocytes reduced TIMP3 mRNA levels by 75% (n=3, p<0·0001). Hydroxyproline release by differentiating pre-adipocytes was 2·3 times greater than that by control-treated cells (mean 5·66 μg/mL [SD 0·77] vs 2·45 [0·36], p<0·0001) indicating greater collagen I degradation. TNF alpha reduced TIMP3 mRNA levels by 66% in in-vitro-differentiated adipocytes (n=3, p<0·0001); reduced TIMP3 expression was confirmed by western blot. Shedding of soluble DLK1 (sDLK1) by pre-adipocytes was increased by TNF alpha and by overexpression of adenovirally delivered TIMP3 compared with control conditions, as confirmed by immunoblot (n=3). Addition of recombinant human sDLK1 (500 pM) to pre-adipocyte cultures reduced adipogenesis, as assessed by oil red O staining (n=2). INTERPRETATION We have shown that TIMP3 is downregulated in adipogenesis, and by inflammatory signals in adipocytes. Furthermore, TIMP3 modulates sDLK1 shedding and collagen I degradation. TIMP3 is known to inhibit ADAM17 (DLK1 sheddase) and MMP14 (implicated in extracellular matrix turnover). TIMP3 might therefore integrate inflammatory signals with adipose remodelling. Subversion of remodelling pathways by chronic adipose inflammation might lead to maladaptive adipose expansion and metabolically unhealthy obesity. FUNDING British Heart Foundation, Diabetes Research and Wellness Foundation Open Funding 2011.

ATP Evokes Ca2+ Responses and CXCL5 Secretion via P2X4 Receptor Activation in Human Monocyte-Derived Macrophages

Leukocytes sense extracellular ATP, a danger-associated molecular pattern, released during cellular stress and death, via activation of cell surface P2X and P2Y receptors. Here, we investigate P2 receptor expression in primary human monocyte-derived macrophages and receptors that mediate ATP-evoked intracellular [Ca2+]i signals and cytokine production in response to ATP concentrations that exclude P2X7 receptor activation. Expression of P2X1, P2X4, P2X5, P2X7, P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, and P2Y13 was confirmed by quantitative RT-PCR and immunocytochemistry. ATP elicited intracellular Ca2+ responses in a concentration-dependent fashion (EC50 = 11.4 ± 2.9 μM, n = 3). P2Y11 and P2Y13 activations mediated the amplitude of [Ca2+]i response, whereas P2X4 activation, but not P2X1 or P2X7, determined the duration of Ca2+ response during a sustained phase. ATP mediated gene induction of CXCL5, a proinflammatory chemokine. P2X4 antagonism (PSB-12062 or BX430) inhibited ATP-mediated induction of CXCL5 gene expression and secretion of CXCL5 by primary macrophage. Inhibition of CXCL5 secretion by P2X4 antagonists was lost in the absence of extracellular Ca2+. Reciprocally, positive allosteric modulation of P2X4 (ivermectin) augmented ATP-mediated CXCL5 secretion. P2X7, P2Y11, or P2Y13 receptor did not contribute to CXCL5 secretion. Together, the data reveals a role for P2X4 in determining the duration of ATP-evoked Ca2+ responses and CXCL5 secretion in human primary macrophage.

Hypercalcaemia – presentation and management

Hypercalcaemia is a common disorder normally caused by primary hyperparathyroidism (PHPT) or malignancy. A proportion of cases present as an emergency, which carries a significant mortality. Emergency management of hypercalcaemia is based on intravenous rehydration with normal saline but when this is inadequate, bisphosphonate therapy is used; more recently the novel anti-resorbtive agent denosumab has been shown to have a useful role in treatment. It is estimated that up to 10% of all cases of PHPT presenting under the age of 45 years have an underlying genetic predisposition; nine potentially causative genes are now recognised and may be screened in routine clinical practice. Although parathyroidectomy is the only curative treatment for PHPT, this is indicated in a minority of cases. Many cases can be adequately managed conservatively and guidance from the 4th international workshop on the management of asymptomatic PHPT has recently been updated in a consensus statement.

Discordance in glycemic categories and regression to normality at baseline in 10,000 people in a Type 2 diabetes prevention trial

The world diabetes population quadrupled between 1980 and 2014 to 422 million and the enormous impact of Type 2 diabetes is recognised by the recent creation of national Type 2 diabetes prevention programmes. There is uncertainty about how to correctly risk stratify people for entry into prevention programmes, how combinations of multiple ‘at high risk’ glycemic categories predict outcome, and how the large recently defined ‘at risk’ population based on an elevated glycosylated haemoglobin (HbA1c) should be managed. We identified all 141,973 people at highest risk of diabetes in our population, and screened 10,000 of these with paired fasting plasma glucose and HbA1c for randomisation into a very large Type 2 diabetes prevention trial. Baseline discordance rate between highest risk categories was 45.6%, and 21.3–37.0% of highest risk glycaemic categories regressed to normality between paired baseline measurements (median 40 days apart). Accurate risk stratification using both fasting plasma glucose and HbA1c data, the use of paired baseline data, and awareness of diagnostic imprecision at diagnostic thresholds would avoid substantial overestimation of the true risk of Type 2 diabetes and the potential benefits (or otherwise) of intervention, in high risk subjects entering prevention trials and programmes.

Constitutive P2Y2 receptor activity regulates basal lipolysis in human adipocytes

ABSTRACT White adipocytes are key regulators of metabolic homeostasis, which release stored energy as free fatty acids via lipolysis. Adipocytes possess both basal and stimulated lipolytic capacity, but limited information exists regarding the molecular mechanisms that regulate basal lipolysis. Here, we describe a mechanism whereby autocrine purinergic signalling and constitutive P2Y2 receptor activation suppresses basal lipolysis in primary human in vitro-differentiated adipocytes. We found that human adipocytes possess cytoplasmic Ca2+ tone due to ATP secretion and constitutive P2Y2 receptor activation. Pharmacological antagonism or knockdown of P2Y2 receptors increases intracellular cAMP levels and enhances basal lipolysis. P2Y2 receptor antagonism works synergistically with phosphodiesterase inhibitors in elevating basal lipolysis, but is dependent upon adenylate cyclase activity. Mechanistically, we suggest that the increased Ca2+ tone exerts an anti-lipolytic effect by suppression of Ca2+-sensitive adenylate cyclase isoforms. We also observed that acute enhancement of basal lipolysis following P2Y2 receptor antagonism alters the profile of secreted adipokines leading to longer-term adaptive decreases in basal lipolysis. Our findings demonstrate that basal lipolysis and adipokine secretion are controlled by autocrine purinergic signalling in human adipocytes. Summary: Here, using pharmacological antagonism or knockdown of P2Y2 receptors, we show that basal lipolysis and adipokine secretion are controlled by autocrine purinergic signalling via P2Y2 receptors in human adipocytes.

MicroRNA expression in a phosphaturic mesenchymal tumour

Phosphaturic mesenchymal tumours are a heterogeneous set of bone and soft tissue neoplasms that can cause a number of paraneoplastic syndromes such as tumour induced osteomalacia. The term phosphaturic comes from the common finding that these tumours secrete high levels of fibroblast growth factor 23 which causes renal phosphate wasting leading to hypophosphatemia. Phosphaturic mesenchymal tumours are rare and diagnosis is difficult. A very active 68 year old male presented with bone pain and muscle weakness. He was hypophosphataemic and total alkaline phosphatase was markedly elevated. The patient was placed on vitamin D supplementation but his condition progressed. In the fifth year of presentation the patient required the use of a wheelchair and described “explosive” bone pain on physical contact. Serum 1,25 dihydroxyvitamin D was low and serum fibroblast growth factor 23 was significantly elevated, raising suspicion of a phosphaturic mesenchymal tumour. A lesion was detected in his left femoral head and the patient underwent a total hip replacement. The patient displayed a rapid improvement to his condition and during a three year follow up period he returned to an active lifestyle. As molecular testing may help provide a robust diagnosis and is particularly useful in rare diseases we took a next generation sequencing approach to identify a differential expression of small RNAs in the resected tumour. Small RNAs are non-coding RNA molecules that play a key role in regulation of gene expression and can be used as specific biomarkers. We found an upregulation of miR-197. We also found a downregulation of miR-20b, miR-144 and miR-335 which is a small RNA profile typical of osteosarcoma. MiR-21, the most frequently upregulated microRNA in cancer, was downregulated. We conclude that the specific small RNA profile is typical of osteosarcoma except for the downregulation of oncogenic miR-21. Transcriptional plasticity of miR-197, which is computationally predicted to target fibroblast growth factor 23 messenger RNA, may be upregulated in a cellular effort to correct the ectopic expression of the protein.

The role of metalloproteinases and their tissue inhibitors in adipose tissue remodelling and whole-body lipid distribution: a cross-sectional clinical study

BACKGROUND Metabolically unhealthy obesity is associated with insulin resistance. Dysfunctional adipose tissue remodelling might explain features of this disorder, such as chronic white adipose tissue inflammation, adipocyte hypertrophy, and ectopic lipid deposition. Metalloproteinases and their tissue inhibitors (TIMPs) have been implicated in human adipose tissue remodelling. In a cross-sectional study, we investigated the association of adipose metalloproteinase and TIMP expression with whole-body lipid distribution and insulin resistance. METHODS Healthy women undergoing elective surgery donated fasting blood samples (for calculation of homoeostasis model assessment of insulin resistance [HOMA2-IR], the primary outcome). At operation 2 cm(3) biopsy samples of subcutaneous and visceral adipose tissue were obtained. 1 cm(3) was fixed, paraffin-embedded, and stained for adipocyte size quantification, and RNA was extracted from the remaining tissue for quantitative RT-PCR analysis. The women also underwent whole-body MRI for analysis of fat distribution. FINDINGS 26 women were recruited (mean age 50·3 years, SD 13·1) into five body-mass index categories (18·5-24·9 kg/m(2) [n=12, 46·1%], 25-29·9 [n=6, 23·1%], 30-34·9 [n=3, 11·5%], 35-39·9 [n=3, 11·5%], >40 [n=2, 7·8%]). Mean fasting glucose was 5·29 mmol/L (SD 0·66), mean fasting insulin 71·29 pmol/L (47·72), and mean HOMA2-IR 1·35 (0·91). HOMA2-IR correlated with body-mass index (r=0·73, p<0·0001), subcutaneous and visceral adipose tissue volumes (r=0·94 and r=0·87, respectively; both p<0·0001), and hepatic fat fraction (r=0·57, p=0·013). Visceral adipose tissue MMP14 expression correlated strongly with hepatic fat fraction (r=0·944, p<0·0001), HOMA2-IR (r=0·74, p=0·01), and visceral adipose tissue volume (r=0·74, p=0·036). Subcutaneous adipose tissue TIMP3 expression correlated with subcutaneous adipocyte area (r=0·72, p=0·029), but not with HOMA2-IR (r=-0·53, p=0·062). INTERPRETATION The results suggest that metalloproteinases and TIMPs regulate adipose tissue remodelling and distribution. MMP14 has been implicated in collagen turnover in pre-adipocyte differentiation, whereas TIMP3 may modulate the shedding of DLK1, a regulator of adipogenesis. In our concurrent in-vitro study, we have shown that human adipocytes express metalloproteinases and TIMPs, and that their expression varies with inflammatory stimulation. These proteins might therefore integrate inflammatory signals with dysregulated adipose remodelling in metabolically unhealthy obesity. FUNDING British Heart Foundation, Diabetes Research & Wellness Foundation Open Funding 2011.