[Show abstract][Hide abstract] ABSTRACT: Tumour cells can use strategies that make them resistant to nutrient deprivation to outcompete their neighbours. A key integrator of the cell's responses to starvation and other stresses is amino-acid-dependent mechanistic target of rapamycin complex 1 (mTORC1). Activation of mTORC1 on late endosomes and lysosomes is facilitated by amino-acid transporters within the solute-linked carrier 36 (SLC36) and SLC38 families. Here, we analyse the functions of SLC36 family member, SLC36A4, otherwise known as proton-assisted amino-acid transporter 4 (PAT4), in colorectal cancer. We show that independent of other major pathological factors, high PAT4 expression is associated with reduced relapse-free survival after colorectal cancer surgery. Consistent with this, PAT4 promotes HCT116 human colorectal cancer cell proliferation in culture and tumour growth in xenograft models. Inducible knockdown in HCT116 cells reveals that PAT4 regulates a form of mTORC1 with two distinct properties: first, it preferentially targets eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), and second, it is resistant to rapamycin treatment. Furthermore, in HCT116 cells two non-essential amino acids, glutamine and serine, which are often rapidly metabolised by tumour cells, regulate rapamycin-resistant mTORC1 in a PAT4-dependent manner. Overexpressed PAT4 is also able to promote rapamycin resistance in human embryonic kidney-293 cells. PAT4 is predominantly associated with the Golgi apparatus in a range of cell types, and in situ proximity ligation analysis shows that PAT4 interacts with both mTORC1 and its regulator Rab1A on the Golgi. These findings, together with other studies, suggest that differentially localised intracellular amino-acid transporters contribute to the activation of alternate forms of mTORC1. Furthermore, our data predict that colorectal cancer cells with high PAT4 expression will be more resistant to depletion of serine and glutamine, allowing them to survive and outgrow neighbouring normal and tumorigenic cells, and potentially providing a new route for pharmacological intervention.Oncogene advance online publication, 5 October 2015; doi:10.1038/onc.2015.363.
[Show abstract][Hide abstract] ABSTRACT: Hypoxia leads to the stabilisation of the hypoxia-inducible factor (HIF) transcription factor that drives the expression of target genes including microRNAs (miRNAs). MicroRNAs are known to regulate many genes involved in tumourigenesis. The aim of this study was to identify hypoxia-regulated miRNAs (HRMs) in bladder cancer and investigate their functional significance.
Bladder cancer cell lines were exposed to normoxic and hypoxic conditions and interrogated for the expression of 384 miRNAs by qPCR. Functional studies were carried out using siRNA-mediated gene knockdown and chromatin immunoprecipitations. Apoptosis was quantified by annexin V staining and flow cytometry.
The HRM signature for NMI bladder cancer lines includes miR-210, miR-193b, miR-145, miR-125-3p, miR-708 and miR-517a. The most hypoxia-upregulated miRNA was miR-145. The miR-145 was a direct target of HIF-1α and two hypoxia response elements were identified within the promoter region of the gene. Finally, the hypoxic upregulation of miR-145 contributed to increased apoptosis in RT4 cells.
We have demonstrated the hypoxic regulation of a number of miRNAs in bladder cancer. We have shown that miR-145 is a novel, robust and direct HIF target gene that in turn leads to increased cell death in NMI bladder cancer cell lines.British Journal of Cancer advance online publication, 21 July 2015; doi:10.1038/bjc.2015.203 www.bjcancer.com.
British Journal of Cancer 07/2015; 113(4). DOI:10.1038/bjc.2015.203 · 4.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Metronomic chemotherapy is the protracted, dense administration of low sub-toxic doses of chemotherapy, to inhibit tumor angiogenesis. Vinorelbine is an orally bioavailable vinca alkaloid shown to be useable for metronomic administration. In clinical trials, metronomic vinorelbine has been demonstrated to generate sustainable antitumor efficacy at low nanomolar (nM) concentrations with negligible toxicity. We sought to determine whether the clinically relevant metronomic concentration of vinorelbine is anti-angiogenic in vitro and whether hypoxia, often induced by anti-angiogenic therapy, modifies its effectiveness. We found that the metronomic concentration of 10 nM vinorelbine inhibited human umbilical vein endothelial cell (HUVEC) proliferation, migration, tube formation and sprouting. Severe hypoxia, did not affect the inhibitory effect of metronomic vinorelbine on migration, tube formation and sprouting. However, severe hypoxia reduced its anti-proliferative effect by decreasing its ability to induce G2/M block as it shifted the cell population to the G1 phase and decreased the fraction of the cells in the DNA synthesis S phase. Furthermore, the pro-apoptotic effects of 10 nM vinorelbine were also decreased. Metronomic vinorelbine decreased the Bcl-2/Bax ratio in normoxia whereas the ratio was reduced in severe hypoxia but unaltered by vinorelbine treatment. Akt signals to an anti-apoptotic pathway and we demonstrated that the Akt inhibitor V reversed the protective effect of severe hypoxia. Thus, we provide evidence for the anti-angiogenic basis of metronomic vinorelbine and we show that severe hypoxia mediates resistance to its anti-proliferative effect on endothelial cells. Akt warrants further investigation as a potential target to circumvent this hypoxic resistance.
International Journal of Oncology 06/2015; 47(2). DOI:10.3892/ijo.2015.3059 · 3.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Oxygen and nutrient limitation are common features of the tumor microenvironment and are associated with cancer progression and induction of metastasis. The inefficient vascularization of tumor tissue also limits the penetration of other serum-derived factors, such as lipids and lipoproteins, which can be rate limiting for cell proliferation and survival. Here we have investigated the effect of hypoxia and serum deprivation on sterol regulatory element-binding protein (SREBP) activity and the expression of lipid metabolism genes in human glioblastoma multiforme (GBM) cancer cells. We found that SREBP transcriptional activity was induced by serum depletion both in normoxic and hypoxic cells and that activation of SREBP was required to maintain the expression of fatty acid and cholesterol metabolism genes under hypoxic conditions. Moreover, expression of stearoyl-CoA desaturase, the enzyme required for the generation of mono-unsaturated fatty acids, and fatty acid-binding protein 7, a regulator of glioma stem cell function, was strongly dependent on SREBP function. Inhibition of SREBP function blocked lipid biosynthesis in hypoxic cancer cells and impaired cell survival under hypoxia and in a three-dimensional spheroid model. Finally, gene expression analysis revealed that SREBP defines a gene signature that is associated with poor survival in glioblastoma.Oncogene advance online publication, 26 January 2015; doi:10.1038/onc.2014.439.
[Show abstract][Hide abstract] ABSTRACT: Hypoxia is a common feature of locally advanced breast cancers that is associated with increased metastasis and poorer survival. Stabilisation of hypoxia-inducible factor-1α (HIF1α) in tumours causes transcriptional changes in numerous genes that function at distinct stages of the metastatic cascade. We demonstrate that expression of RIOK3 (RIght Open reading frame kinase 3) was increased during hypoxic exposure in an HIF1α-dependent manner. RIOK3 was localised to distinct cytoplasmic aggregates in normoxic cells and underwent redistribution to the leading edge of the cell in hypoxia with a corresponding change in the organisation of the actin cytoskeleton. Depletion of RIOK3 expression caused MDA-MB-231 to become elongated and this morphological change was due to a loss of protraction at the trailing edge of the cell. This phenotypic change resulted in reduced cell migration in two-dimensional cultures and inhibition of cell invasion through three-dimensional extracellular matrix. Proteomic analysis identified interactions of RIOK3 with actin and several actin-binding factors including tropomyosins (TPM3 and TPM4) and tropomodulin 3. Depletion of RIOK3 in cells resulted in fewer and less organised actin filaments. Analysis of these filaments showed reduced association of TPM3, particularly during hypoxia, suggesting that RIOK3 regulates actin filament specialisation. RIOK3 depletion reduced the dissemination of MDA-MB-231 cells in both a zebrafish model of systemic metastasis and a mouse model of pulmonary metastasis. These findings demonstrate that RIOK3 is necessary for maintaining actin cytoskeletal organisation required for migration and invasion, biological processes that are necessary for hypoxia-driven metastasis.Oncogene advance online publication, 8 December 2014; doi:10.1038/onc.2014.396.
[Show abstract][Hide abstract] ABSTRACT: Purpose:
To assess whether the combination of letrozole, metronomic cyclophosphamide and sorafenib (LCS) is well tolerated and shows activity in primary breast cancer (BC).
Thirteen oestrogen receptor-positive, postmenopausal, T2-4, N0-1 BC patients received the LCS combination for 6 months. In these patients we examined the pharmacokinetics of sorafenib and cyclophosphamide, toxicity of the regimen, the clinical response to therapy and changes in the levels of biologically relevant biomarkers.
Adequate plasma concentrations of sorafenib were achieved in patients when it was dosed in combination with L+C. The mean plasma concentrations of C were consistently lower following administration of LCS, compared with administration of L+C only. The most common drug-related grade 3/4 adverse events were skin rash (69.3%), hand-foot skin reaction (69.3%) and diarrhoea (46.1%). According to RECIST Criteria, a clinical complete response was observed in 6 of 13 patients. A significant reduction in tumour size, evaluated with MRI, was also observed between baseline and 14 days of treatment in all 13 patients (P=0.005). A significant reduction in SUV uptake, measured by (18)FDG-PET/CT, was observed in all patients between baseline and 30 days of treatment (P=0.015) and between baseline and definitive surgery (P=0.0002). Using modified CT Criteria, a response was demonstrated in 8 out of 10 evaluable patients at 30 days and in 11 out of 13 evaluable patients at the definitive surgery. A significant reduction in Ki67 expression was observed in all patients at day 14 compared with baseline (P<0.00001) and in 9 out of 13 patients at the definitive surgery compared with baseline (P<0.03). There was also a significant suppression of CD31 and VEGF-A expression in response to treatment (P=0.01 and P=0.007, respectively).
The LCS combination is feasible and tolerable. The tumour response and target biomarker modulation indicate that the combination is clinically and biologically active.
British Journal of Cancer 12/2014; 112(1). DOI:10.1038/bjc.2014.563 · 4.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Activation of cellular transcriptional responses, mediated by hypoxia-inducible factor (HIF), is common in many types of cancer, and generally confers a poor prognosis. Known to induce many hundreds of protein-coding genes, HIF has also recently been shown to be a key regulator of the non-coding transcriptional response. Here, we show that NEAT1 long non-coding RNA (lncRNA) is a direct transcriptional target of HIF in many breast cancer cell lines and in solid tumors. Unlike previously described lncRNAs, NEAT1 is regulated principally by HIF-2 rather than by HIF-1. NEAT1 is a nuclear lncRNA that is an essential structural component of paraspeckles and the hypoxic induction of NEAT1 induces paraspeckle formation in a manner that is dependent upon both NEAT1 and on HIF-2. Paraspeckles are multifunction nuclear structures that sequester transcriptionally active proteins as well as RNA transcripts that have been subjected to adenosine-to-inosine (A-to-I) editing. We show that the nuclear retention of one such transcript, F11R (also known as junctional adhesion molecule 1, JAM1), in hypoxia is dependent upon the hypoxic increase in NEAT1, thereby conferring a novel mechanism of HIF-dependent gene regulation. Induction of NEAT1 in hypoxia also leads to accelerated cellular proliferation, improved clonogenic survival and reduced apoptosis, all of which are hallmarks of increased tumorigenesis. Furthermore, in patients with breast cancer, high tumor NEAT1 expression correlates with poor survival. Taken together, these results indicate a new role for HIF transcriptional pathways in the regulation of nuclear structure and that this contributes to the pro-tumorigenic hypoxia-phenotype in breast cancer.Oncogene advance online publication, 24 November 2014; doi:10.1038/onc.2014.378.
[Show abstract][Hide abstract] ABSTRACT: Stable isotopes are ideal labels for studying biological processes because they have little or no effect on the biochemical properties of target molecules. The NanoSIMS is a tool that can image the distribution of stable isotope labels with up to 50 nm spatial resolution and with good quantitation. This combination of features has enabled several groups to undertake significant experiments on biological problems in the last decade. Combining the NanoSIMS with other imaging techniques also enables us to obtain not only chemical information but also the structural information needed to understand biological processes. This article describes the methodologies that we have developed to correlate atomic force microscopy and backscattered electron imaging with NanoSIMS experiments to illustrate the imaging of stable isotopes at molecular, cellular, and tissue scales. Our studies make it possible to address 3 biological problems: (1) the interaction of antimicrobial peptides with membranes; (2) glutamine metabolism in cancer cells; and (3) lipoprotein interactions in different tissues.
[Show abstract][Hide abstract] ABSTRACT: Background:
Stratification of patients for treatment of ductal carcinoma in situ (DCIS) is suboptimal, with high systemic overtreatment rates.
A training set of 95 tumours from women with pure DCIS were immunostained for proteins involved in cell survival, hypoxia, growth factor and hormone signalling. A generalised linear regression with regularisation and variable selection was applied to a multiple covariate Cox survival analysis with recurrence-free survival 10-fold cross-validation and leave-one-out iterative approach were used to build and test the model that was validated using an independent cohort of 58 patients with pure DCIS. The clinical role of a COX-2-targeting agent was then tested in a proof-of-concept neoadjuvant randomised trial in ER-positive DCIS treated with exemestane 25 mg day−1±celecoxib 800 mg day−1.
The COX-2 expression was an independent prognostic factor for early relapse in the training (HR 37.47 (95% CI: 5.56–252.74) P=0.0001) and independent validation cohort (HR 3.9 (95% CI: 1.8–8.3) P=0.002). There was no significant interaction with other clinicopathological variables. A statistically significant reduction of Ki-67 expression after treatment with exemestane±celecoxib was observed (P<0.02) with greater reduction in the combination arm (P<0.004). Concomitant reduction in COX-2 expression was statistically significant in the exemestane and celecoxib arm (P<0.03) only.
In patients with DCIS, COX-2 may predict recurrence, aiding clinical decision making. A combination of an aromatase inhibitor and celecoxib has significant biological effect and may be integrated into treatment of COX2-positive DCIS at high risk of recurrence.
British Journal of Cancer 05/2014; 111(1). DOI:10.1038/bjc.2014.236 · 4.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background:
Although Trastuzumab has improved survival of HER2+ breast cancer patients, resistance to the agent pre-exists or develops through the course of therapy. Here we show that a specific metabolism and autophagy-related cancer cell phenotype relates to resistance of HER2+ breast cancer to Trastuzumab and chemotherapy.
Twenty-eight patients with locally advanced primary breast cancer were prospectively scheduled to received one cycle of Trastuzumab followed by a new biopsy on day 21, followed by taxol/Trastuzumab chemotherapy for four cycles before surgery. FDG PET/CT scan was used to monitor tumour response. Tissue samples were immunohistochemically analysed for metabolism and autophagy markers.
In pre-Trastuzumab biopsies, the LC3A+/HER2+ cell population was correlated with HIF1α expression (P=0.01), while GLUT1 and LC3B expression were correlated with Ki67 proliferation index (P=0.01 and P=0.01, respectively). FDG PET tumour dimensions before therapy were correlated with LC3B expression (P=0.005). Administration of Trastuzumab significantly reduced clinical and PET-detected tumour dimensions (P<0.01). An inverse association of tumour response with the percentage of cells expressing HIF1α at baseline was documented (P=0.01). Administration of Trastuzumab resulted in a decrease of the proliferation index (P=0.004), GLUT1 (P=0.04) and HER2 (P=0.01) expression. In contrast, the percentage of LC3A+/HER2+ cells was increased (P=0.01). High baseline HIF1α expression was the only parameter associated with poorer pathological response to preoperative chemotherapy (P=0.001).
As the HER2+/LC3A+ phenotype, which often overexpresses HIF1α, is a major subpopulation increasing after therapy with Trastuzumab, LC3A- and HIF1α-targeting therapies should be investigated for the augmentation of anti-HER2 therapy efficacy.
British Journal of Cancer 04/2014; 110(9). DOI:10.1038/bjc.2014.196 · 4.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background:
The lipogenic transcription factor carbohydrate response element-binding protein (ChREBP) may play a key role in malignant progression of breast cancer by allowing metabolic adaptations to take place in response to changes in oxygenation.
Immunohistochemical analysis of ChREBP was carried out in human breast tumour tissue microarrays representative of malignant progression from normal breast through to metastatic cancer. The ChREBP protein and mRNA expressions were then analysed in a series of breast cancers for correlative analysis with common and breast-specific hypoxia signatures, and survival.
In invasive ductal carcinoma, ChREBP correlated significantly with mean ‘downregulated' hypoxia scores (r=0.3, P<0.015, n=67) and in two distinct breast progression arrays, ChREBP protein also increased with malignant progression (P<0.001). However, bioinformatic analysis of a large data set (2136 cases) revealed an apparent reversal in the relationship between ChREBP mRNA level and clinical outcome – not only being significantly correlated with increased survival (log rank P<0.001), but also downregulated in malignant tissue compared with adjacent normal tissue.
The ChREBP expression may be reflective of an aerobic metabolic phenotype that may conflict with hypoxia-induced signalling but provide a mechanism for growth at the oxygenated edge of the tumours.
British Journal of Cancer 12/2013; 110(3). DOI:10.1038/bjc.2013.765 · 4.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A breast cancer-selective oncolytic adenovirus was engineered to express antagonists of vascular endothelial growth factor (VEGF) and Notch signaling to combine direct anticancer activity with disruption of tumor-associated angiogenesis. Replication of the parental virus, AdEHE2F, is stimulated by estrogen receptor (ER), E2F1 and hypoxia, and it mediates selective lysis of breast cancer cells in vitro and in vivo. Here, we encoded soluble Flt-1 (sFlt1) and soluble Dll4 (sDll4) under control of the E3 promoter. sFlt1 (the extra-cellular domain of VEGF receptor 1) binds VEGF-A and inhibits stimulation of VEGFR2, decreasing angiogenic stimulus. Conversely, sDll4 (the extracellular domain of Delta-like 4) antagonizes Notch signaling to prevent endothelial maturation. We hypothesized that these agents might show additive or synergistic activity. In vitro, sFlt1 inhibited endothelial cell proliferation and sprouting, whereas sDll4 increased the number of vascular branchpoints. In ER-positive ZR75.1 tumors in vivo AdEHE2F showed the potent direct virotherapy with no augmentation owing to sFlt1 or sDll4; however, in ER-negative MDA-231 tumors efficacy was enhanced by encoding sFlt1 or sDll4, with survival time extending to double that of controls. There was also a dramatic decrease in the total number of tumour blood vessels, as well as the number of perfused vessels, suggesting that improved efficacy reflects combined anti-tumour and anti-vascular effects.Cancer Gene Therapy advance online publication, 12 July 2013; doi:10.1038/cgt.2013.41.
Cancer gene therapy 07/2013; 20(8). DOI:10.1038/cgt.2013.41 · 2.42 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background:
Non-muscle invasive (NMI) bladder cancer is characterised by increased expression and activating mutations of FGFR3. We have previously investigated the role of microRNAs in bladder cancer and have shown that FGFR3 is a target of miR-100. In this study, we investigated the effects of hypoxia on miR-100 and FGFR3 expression, and the link between miR-100 and FGFR3 in hypoxia.
Bladder cancer cell lines were exposed to normoxic or hypoxic conditions and examined for the expression of FGFR3 by quantitative PCR (qPCR) and western blotting, and miR-100 by qPCR. The effect of FGFR3 and miR-100 on cell viability in two-dimensional (2-D) and three-dimensional (3-D) was examined by transfecting siRNA or mimic-100, respectively.
In NMI bladder cancer cell lines, FGFR3 expression was induced by hypoxia in a transcriptional and HIF-1α-dependent manner. Increased FGFR3 was also in part dependent on miR-100 levels, which decreased in hypoxia. Knockdown of FGFR3 led to a decrease in phosphorylation of the downstream kinases mitogen-activated protein kinase (MAPK) and protein kinase B (PKB), which was more pronounced under hypoxic conditions. Furthermore, transfection of mimic-100 also decreased phosphorylation of MAPK and PKB. Finally, knocking down FGFR3 profoundly decreased 2-D and 3-D cell growth, whereas introduction of mimic-100 decreased 3-D growth of cells.
Hypoxia, in part via suppression of miR-100, induces FGFR3 expression in bladder cancer, both of which have an important role in maintaining cell viability under conditions of stress.
British Journal of Cancer 06/2013; 109(1). DOI:10.1038/bjc.2013.240 · 4.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The BJC is owned by Cancer Research UK, a charity dedicated to understanding the causes, prevention and treatment of cancer and to making sure that the best new treatments reach patients in the clinic as quickly as possible. The journal reflects these aims. It was founded more than fifty years ago and, from the start, its far-sighted mission was to encourage communication of the very best cancer research from laboratories and clinics in all countries. The breadth of its coverage, its editorial independence and it consistent high standards, have made BJC one of the world's premier general cancer journals. Its increasing popularity is reflected by a steadily rising impact factor.
British Journal of Cancer 06/2013; 108(11). DOI:10.1038/bjc.2013.259 · 4.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background:
The objective of this study was to determine the optimal scheduling of 2.5 mg daily letrozole in neoadjuvant breast cancer patients to obtain pathological complete response (pathCR) and assess Ki-67 expression as an early predictor of response.
Patients and methods:
This single institution study comprised 120 oestrogen receptor (ER)-positive postmenopausal women with primary breast cancer (clinical stage ⩾T2, N0–1), from three sequential cohorts (cohort A of 40, cohort B of 40 and cohort C of 40 patients, respectively) based on different duration of the neoadjuvant letrozole. Biological markers such as ER, progesterone receptor, HER2 and Ki-67 expression were tested at diagnosis and at definitive surgery.
A total of 89 patients (75.4%) achieved an objective response with 44 (37.3%) clinical CRs and 45 (38.1%) partial responses. The clinical CRs were significantly observed in cohort C (23 out of 40 patients, 57.5%) and B (16 out of 38 patients, 42.1%) compared with cohort A (5 out of 40 patients, 12.5%) (P-value for trend <0.001). Letrozole induced a similar significant reduction in Ki-67 index after treatment in all cohorts. The pathCR rate was significantly more frequent in cohort C (7 out of 40 patients, 17.5%) than in cohort A (1 out of 40 patients, 2.5%) and B (2 out of 40 patients, 5.0%) (P-value for trend <0.04).
One-year neoadjuvant letrozole therapy leads to a higher pathCR rate and may be the optimal length of drug exposure.
British Journal of Cancer 04/2013; 108(8). DOI:10.1038/bjc.2013.151 · 4.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background:
Clear cell renal cancer frequently harbours von Hippel–Lindau (VHL) gene mutations, leading to stabilisation of the hypoxia-inducible factors (HIFs) and expression of their target genes. We investigated HIF-1 and HIF-2 in the regulation of microRNA-210 (miR-210), and its clinical relevance in renal tumours.
RCC4 and 786-O renal cancer cell lines transfected with either an empty vector or functional VHL and incubated in normoxia or hypoxia were examined for miR-210 expression. Hypoxia-inducible factor siRNAs were used to examine their regulation of miR-210. Seventy-one clear cell renal tumours were sequenced for VHL mutations. Expression of miR-210, VHL, CA9, ISCU and Ki-67 were determined by immunohistochemistry and qRT–PCR.
In addition to HIF-1 regulating miR-210 in renal cancer, HIF-2 can regulate this microRNA in the absence of HIF-1. MicroRNA-210 is upregulated in renal cancer compared with normal renal cortex tissue. MicroRNA-210 correlates negatively with its gene target ISCU at the protein and mRNA level. MicroRNA-210 correlated with positive outcome variables and negatively with Ki-67.
We provide further evidence of miR-210 activity in vivo, and show that high miR-210 expression is associated with better clinico-pathological prognostic factors.
British Journal of Cancer 02/2013; 108(5). DOI:10.1038/bjc.2013.56 · 4.84 Impact Factor