Publications by authors named "Puthen V Jithesh"

22 Publications

  • Page 1 of 1

Integrated omics profiling reveals novel patterns of epigenetic programming in cancer-associated myofibroblasts.

Carcinogenesis 2019 06;40(4):500-512

Department of Cellular and Molecular Physiology, University of Liverpool, Liverpool, UK.

There is increasing evidence that stromal myofibroblasts play a key role in the tumour development however, the mechanisms by which they become reprogrammed to assist in cancer progression remain unclear. As cultured cancer-associated myofibroblasts (CAMs) retain an ability to enhance the proliferation and migration of cancer cells in vitro, it is possible that epigenetic reprogramming of CAMs within the tumour microenvironment may confer long-term pro-tumourigenic changes in gene expression. This study reports the first comparative multi-omics analysis of cancer-related changes in gene expression and DNA methylation in primary myofibroblasts derived from gastric and oesophageal tumours. In addition, we identify novel CAM-specific DNA methylation signatures, which are not observed in patient-matched adjacent tissue-derived myofibroblasts, or corresponding normal tissue-derived myofibroblasts. Analysis of correlated changes in DNA methylation and gene expression shows that different patterns of gene-specific DNA methylation have the potential to confer pro-tumourigenic changes in metabolism, cell signalling and differential responses to hypoxia. These molecular signatures provide new insights into potential mechanisms of stromal reprogramming in gastric and oesophageal cancer, while also providing a new resource to facilitate biomarker identification and future hypothesis-driven studies into mechanisms of stromal reprogramming and tumour progression in solid tumours.
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http://dx.doi.org/10.1093/carcin/bgz001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6556705PMC
June 2019

The Unfolded Protein Response: A Novel Therapeutic Target for Poor Prognostic Mutant Colorectal Cancer.

Mol Cancer Ther 2018 06 26;17(6):1280-1290. Epub 2018 Feb 26.

Drug Resistance Group, Centre for Cancer Research and Cell Biology, School of Medicine, Dentistry and Biomedical Science, Queen's University Belfast, Belfast, United Kingdom.

mutations occur in ∼10% of colorectal cancer cases, are associated with poor survival, and have limited responses to BRAF/MEK inhibition with or without EGFR inhibition. There is an unmet need to understand the biology of poor prognostic MT colorectal cancer. We have used differential gene expression and pathway analyses of untreated stage II and stage III MT (discovery set: = 31; validation set: = 26) colorectal cancer, and an siRNA screen to characterize the biology underpinning the MT subgroup with poorest outcome. These analyses identified the unfolded protein response (UPR) as a novel and druggable pathway associated with the MT colorectal cancer subgroup with poorest outcome. We also found that oncogenic drives endoplasmic reticulum (ER) stress and UPR pathway activation through MEK/ERK. Furthermore, inhibition of GRP78, the master regulator of the UPR, using siRNA or small molecule inhibition, resulted in acute ER stress and apoptosis, in particular in MT colorectal cancer cells. In addition, dual targeting of protein degradation using combined Carfilzomib (proteasome inhibitor) and ACY-1215 (HDAC6-selective inhibitor) treatment resulted in marked accumulation of protein aggregates, acute ER stress, apoptosis, and therapeutic efficacy in MT and xenograft models. Mechanistically, we found that the apoptosis following combined Carfilzomib/ACY-1215 treatment is mediated through increased CHOP expression. Taken together, our findings indicate that oncogenic induces chronic ER stress and that inducers of acute ER stress could be a novel treatment strategy for poor prognostic MT colorectal cancer. .
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http://dx.doi.org/10.1158/1535-7163.MCT-17-0603DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5985931PMC
June 2018

Accelerating next generation sequencing data analysis with system level optimizations.

Sci Rep 2017 08 22;7(1):9058. Epub 2017 Aug 22.

Biomedical Informatics, Research Branch, Sidra Medical and Research Center, Post Box No. 26999, Doha, Qatar.

Next generation sequencing (NGS) data analysis is highly compute intensive. In-memory computing, vectorization, bulk data transfer, CPU frequency scaling are some of the hardware features in the modern computing architectures. To get the best execution time and utilize these hardware features, it is necessary to tune the system level parameters before running the application. We studied the GATK-HaplotypeCaller which is part of common NGS workflows, that consume more than 43% of the total execution time. Multiple GATK 3.x versions were benchmarked and the execution time of HaplotypeCaller was optimized by various system level parameters which included: (i) tuning the parallel garbage collection and kernel shared memory to simulate in-memory computing, (ii) architecture-specific tuning in the PairHMM library for vectorization, (iii) including Java 1.8 features through GATK source code compilation and building a runtime environment for parallel sorting and bulk data transfer (iv) the default 'on-demand' mode of CPU frequency is over-clocked by using 'performance-mode' to accelerate the Java multi-threads. As a result, the HaplotypeCaller execution time was reduced by 82.66% in GATK 3.3 and 42.61% in GATK 3.7. Overall, the execution time of NGS pipeline was reduced to 70.60% and 34.14% for GATK 3.3 and GATK 3.7 respectively.
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http://dx.doi.org/10.1038/s41598-017-09089-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5567265PMC
August 2017

Gene expression profiling in bladder cancer identifies potential therapeutic targets.

Int J Oncol 2017 Apr 2;50(4):1147-1159. Epub 2017 Mar 2.

School of Cancer Sciences, University of Birmingham, Birmingham B15 2TT, UK.

Despite advances in management, bladder cancer remains a major cause of cancer related complications. Characterisation of gene expression patterns in bladder cancer allows the identification of pathways involved in its pathogenesis, and may stimulate the development of novel therapies targeting these pathways. Between 2004 and 2005, cystoscopic bladder biopsies were obtained from 19 patients and 11 controls. These were subjected to whole transcript-based microarray analysis. Unsupervised hierarchical clustering was used to identify samples with similar expression profiles. Hypergeometric analysis was used to identify canonical pathways and curated networks having statistically significant enrichment of differentially expressed genes. Osteopontin (OPN) expression was validated by immunohistochemistry. Hierarchical clustering defined signatures, which differentiated between cancer and healthy tissue, muscle-invasive or non-muscle invasive cancer and healthy tissue, grade 1 and grade 3. Pathways associated with cell cycle and proliferation were markedly upregulated in muscle-invasive and grade 3 cancers. Genes associated with the classical complement pathway were downregulated in non-muscle invasive cancer. Osteopontin was markedly overexpressed in invasive cancer compared to healthy tissue. The present study contributes to a growing body of work on gene expression signatures in bladder cancer. The data support an important role for osteopontin in bladder cancer, and identify several pathways worthy of further investigation.
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http://dx.doi.org/10.3892/ijo.2017.3893DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5363876PMC
April 2017

Genome-wide Regulatory Roles of the C2H2-type Zinc Finger Protein ZNF764 on the Glucocorticoid Receptor.

Sci Rep 2017 01 31;7:41598. Epub 2017 Jan 31.

Division of Translational Medicine, Sidra Medical and Research Center, Doha 26999, Qatar.

The C2H2-type zinc finger protein ZNF764 acts as an enhancer for several steroid hormone receptors, and haploinsufficiency of this gene may be responsible for tissue resistance to multiple steroid hormones including glucocorticoids observed in a patient with 16p11.2 microdeletion. We examined genome-wide regulatory actions of ZNF764 on the glucocorticoid receptor (GR) in HeLa cells as a model system. ZNF764- and GR-binding sites demonstrated similar distribution in various genomic features. They positioned predominantly around 50-500 kbs from the transcription start sites of their nearby genes, and were closely localized with each other, overlapping in ~37% of them. ZNF764 demonstrated differential on/off effects on GR-binding and subsequent mRNA expression: some genes were highly dependent on the presence/absence of ZNF764, but others were not. Pathway analysis revealed that these 3 gene groups were involved in distinct cellular activities. ZNF764 physically interacted with GR at ligand-binding domain through its KRAB domain, and both its physical interaction to GR and zinc finger domain appear to be required for ZNF764 to regulate GR transcriptional activity. Thus, ZNF764 is a cofactor directing GR transcriptional activity toward specific biologic pathways by changing GR binding and transcriptional activity on the glucocorticoid-responsive genes.
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http://dx.doi.org/10.1038/srep41598DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5282477PMC
January 2017

AKT1 has dual actions on the glucocorticoid receptor by cooperating with 14-3-3.

Mol Cell Endocrinol 2017 01 4;439:431-443. Epub 2016 Oct 4.

Division of Translational Medicine, Sidra Medical and Research Center, Out Patient Clinic, PO Box 26999, Al Luqta Street, Education City North Campus, Doha, Qatar; Program in Reproductive and Adult Endocrinology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bldg. 10, CRC, Rm 1-3140, 10 Center Drive MSC 1109, Bethesda, MD 20892, USA. Electronic address:

Glucocorticoids are important therapeutic compounds for acute lymphoblastic leukemia (ALL). AKT1 or the protein kinase B is frequently activated in ALL, and contributes to the development of glucocorticoid resistance. We examined impact of AKT1 on glucocorticoid receptor (GR)-induced transcriptional activity in cooperation with phospho-serine/threonine-binding protein 14-3-3. AKT1 has two distinct actions on GR transcriptional activity, one through segregation of GR in the cytoplasm by phosphorylating GR at Ser-134 and subsequent association of 14-3-3, and the other through direct modulation of GR transcriptional activity in the nucleus. For the latter, AKT1 and 14-3-3 are attracted to DNA-bound GR, accompanied by AKT1-dependent p300 phosphorylation, H3S10 phosphorylation and H3K14 acetylation at the DNA site. These two actions of AKT1 regulate distinct sets of glucocorticoid-responsive genes. Our results suggest that specific inhibition of the AKT1/14-3-3 activity on the cytoplasmic retention of GR may be a promising target for treating glucocorticoid resistance observed in ALL.
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http://dx.doi.org/10.1016/j.mce.2016.10.002DOI Listing
January 2017

Ran GTPase promotes cancer progression via Met recepto-rmediated downstream signaling.

Oncotarget 2016 11;7(46):75854-75864

Institute of Cancer Therapeutics, University of Bradford, Bradford, West Yorkshire, UK.

It has been shown previously that cancer cells with an activated oncogenic pathway, including Met activation, require Ran for growth and survival.Here, we show that knockdown of Ran leads to a reduction of Met receptor expression in several breast and lung cancer cell lines. This, in turn suppressed HGF expression and the Met-mediated activation of the Akt pathway, as well as cell adhesion, migration, and invasion. In a cell line model where Met amplification has previously been shown to contribute to gefitinib resistance, Ran knockdown sensitized cells to gefitinib-mediated inhibition of Akt and ERK1/2 phosphorylation and consequently reduced cell proliferation. We further demonstrate that Met reduction-mediated by knockdown of Ran, occurs at the post-transcriptional level, probably via a matrix metalloproteinase. Moreover, the level of immunoreactive Ran and Met are positively associated in human breast cancer specimens, suggesting that a high level of Ran may be a pre-requisite for Met overexpression. Interestingly, a high level of immunoreactive Ran dictates the prognostic significance of Met, indicating that the co-overexpression of Met and Ran may be associated with cancer progression and could be used in combination as a prognostic indicator.
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http://dx.doi.org/10.18632/oncotarget.12420DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5342783PMC
November 2016

KDM6B histone demethylase is an epigenetic regulator of estrogen receptor β expression in human pleural mesothelioma.

Epigenomics 2016 09 16;8(9):1227-38. Epub 2016 Aug 16.

Department of Pharmaceutical Sciences, University of Piemonte Orientale, Lgo Donegani 2, 28100 Novara, Italy.

Aim: To assess the correlation between KDM6B and estrogen receptor β (ERβ) expression in malignant pleural mesothelioma (MPM).

Materials & Methods: We evaluated gene expression by in silico analysis of microarray data, real-time PCR and western blot in MPM tumors and cell lines.

Results & Conclusion: We report a strong positive correlation between the expression of KDM6B and ERβ in MPM tumors and cell lines. We describe that, in hypoxia, the HIF2α-KDM6B axis induces an epithelioid morphology and ERβ expression in biphasic MPM cells with estrogen receptor-negative phenotype. Reduced histone H3K27 tri-methylation confirms KDM6B activity under hypoxic conditions. Importantly, cells treated during reoxygenation with the selective ERβ agonist, KB9520, maintain ERβ expression and the less aggressive phenotype acquired in hypoxia.
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http://dx.doi.org/10.2217/epi-2016-0025DOI Listing
September 2016

Temple-Baraitser Syndrome and Zimmermann-Laband Syndrome: one clinical entity?

BMC Med Genet 2016 Jun 10;17(1):42. Epub 2016 Jun 10.

Laboratory of Genetic Medicine and Immunology, Weill Cornell Medicine-Qatar, Education City, Qatar Foundation, Doha, Qatar.

Background: KCNH1 encodes a voltage-gated potassium channel that is predominantly expressed in the central nervous system. Mutations in this gene were recently found to be responsible for Temple-Baraitser Syndrome (TMBTS) and Zimmermann-Laband syndrome (ZLS).

Methods: Here, we report a new case of TMBTS diagnosed in a Lebanese child. Whole genome sequencing was carried out on DNA samples of the proband and his parents to identify mutations associated with this disease. Sanger sequencing was performed to confirm the presence of detected variants.

Results: Whole genome sequencing revealed three missense mutations in TMBTS patient: c.1042G > A in KCNH1, c.2131 T > C in STK36, and c.726C > A in ZNF517. According to all predictors, mutation in KCNH1 is damaging de novo mutation that results in substitution of Glycine by Arginine, i.e., p.(Gly348Arg). This mutation was already reported in a patient with ZLS that could affect the connecting loop between helices S4-S5 of KCNH1 with a gain of function effect.

Conclusions: Our findings demonstrate that KCNH1 mutations cause TMBTS and expand the mutational spectrum of KCNH1 in TMBTS. In addition, all cases of TMBTS were reviewed and compared to ZLS. We suggest that the two syndromes are a continuum and that the variability in the phenotypes is the result of the involvement of genetic modifiers.
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http://dx.doi.org/10.1186/s12881-016-0304-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4901505PMC
June 2016

Analysis of Gene Expression in 3D Spheroids Highlights a Survival Role for ASS1 in Mesothelioma.

PLoS One 2016 16;11(3):e0150044. Epub 2016 Mar 16.

Department of Medicine, Pulmonary, San Francisco General Hospital, University of California San Francisco, San Francisco, California, United States of America.

To investigate the underlying causes of chemoresistance in malignant pleural mesothelioma, we have studied mesothelioma cell lines as 3D spheroids, which acquire increased chemoresistance compared to 2D monolayers. We asked whether the gene expression of 3D spheroids would reveal mechanisms of resistance. To address this, we measured gene expression of three mesothelioma cell lines, M28, REN and VAMT, grown as 2D monolayers and 3D spheroids. A total of 209 genes were differentially expressed in common by the three cell lines in 3D (138 upregulated and 71 downregulated), although a clear resistance pathway was not apparent. We then compared the list of 3D genes with two publicly available datasets of gene expression of 56 pleural mesotheliomas compared to normal tissues. Interestingly, only three genes were increased in both 3D spheroids and human tumors: argininosuccinate synthase 1 (ASS1), annexin A4 (ANXA4) and major vault protein (MVP); of these, ASS1 was the only consistently upregulated of the three genes by qRT-PCR. To measure ASS1 protein expression, we stained 2 sets of tissue microarrays (TMA): one with 88 pleural mesothelioma samples and the other with additional 88 pleural mesotheliomas paired with matched normal tissues. Of the 176 tumors represented on the two TMAs, ASS1 was expressed in 87 (50%; staining greater than 1 up to 3+). For the paired samples, ASS1 expression in mesothelioma was significantly greater than in the normal tissues. Reduction of ASS1 expression by siRNA significantly sensitized mesothelioma spheroids to the pro-apoptotic effects of bortezomib and of cisplatin plus pemetrexed. Although mesothelioma is considered by many to be an ASS1-deficient tumor, our results show that ASS1 is elevated at the mRNA and protein levels in mesothelioma 3D spheroids and in human pleural mesotheliomas. We also have uncovered a survival role for ASS1, which may be amenable to targeting to undermine mesothelioma multicellular resistance.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0150044PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4794185PMC
July 2016

Distinct miRNA profiles in normal and gastric cancer myofibroblasts and significance in Wnt signaling.

Am J Physiol Gastrointest Liver Physiol 2016 05 3;310(9):G696-704. Epub 2016 Mar 3.

Departments of Cellular and Molecular Physiology and

Stromal cells influence epithelial function in both health and disease. Myofibroblasts are abundant stromal cells that influence the cellular microenvironment by release of extracellular matrix (ECM) proteins, growth factors, proteases, cytokines, and chemokines. Cancer-associated myofibroblasts (CAMs) differ from adjacent tissue (ATMs) and normal tissue myofibroblasts (NTMs), but the basis of this is incompletely understood. We report now the differential expression of miRNAs in gastric cancer CAMs. MicroRNA arrays identified differences in the miRNA profile in gastric and esophageal NTMs and in CAMs from stomach compared with NTMs. miR-181d was upregulated in gastric CAMs. Analysis of differentially regulated miRNAs indicated an involvement in Wnt signaling. Examination of a microarray data set then identified Wnt5a as the only consistently upregulated Wnt ligand in gastric CAMs. Wnt5a stimulated miR-181d expression, and knockdown of miR-181d inhibited Wnt5a stimulation of CAM proliferation and migration. Analysis of miR-181d targets suggested a role in chemotaxis. Conditioned medium from CAMs stimulated gastric cancer cell (AGS) migration more than that from ATMs, and miR-181d knockdown reduced the effect of CAM-CM on AGS cell migration but had no effect on AGS cell responses to ATM conditioned media. The data suggest that dysregulation of miRNA expression in gastric CAMs, secondary to Wnt5a signaling, accounts at least in part for the effect of CAMs in promoting cancer cell migration.
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http://dx.doi.org/10.1152/ajpgi.00443.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4867324PMC
May 2016

SIRT1 at the crossroads of AKT1 and ERβ in malignant pleural mesothelioma cells.

Oncotarget 2016 Mar;7(12):14366-79

Department of Pharmaceutical Sciences, University of Piemonte Orientale "A. Avogadro", 28100 Novara, Italy.

In this report, we show that malignant pleural mesothelioma (MPM) patients whose tumors express high levels of AKT1 exhibit a significantly worse prognosis, whereas no significant correlation with AKT3 expression is observed. We provide data that establish a phosphorylation independent role of AKT1 in affecting MPM cell shape and anchorage independent cell growth in vitro and highlight the AKT1 isoform-specific nature of these effects.We describe that AKT1 activity is inhibited by the loss of SIRT1-mediated deacetylation and identify, by mass spectrometry, 11 unique proteins that interact with acetylated AKT1.Our data demonstrate a role of the AKT1/SIRT1/FOXM1 axis in the expression of the tumor suppressor ERβ. We further demonstrate an inhibitory feedback loop by ERβ, activated by the selective agonist KB9520, on this axis both in vitro and in vivo.Our data broaden the current knowledge of ERβ and AKT isoform-specific functions that could be valuable in the design of novel and effective therapeutic strategies for MPM.
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http://dx.doi.org/10.18632/oncotarget.7321DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4924721PMC
March 2016

Depletion of Human DNA in Spiked Clinical Specimens for Improvement of Sensitivity of Pathogen Detection by Next-Generation Sequencing.

J Clin Microbiol 2016 Apr 13;54(4):919-27. Epub 2016 Jan 13.

Children's and Women's Health Centre of BC, PHSA, Vancouver, British Columbia, Canada Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada.

Next-generation sequencing (NGS) technology has shown promise for the detection of human pathogens from clinical samples. However, one of the major obstacles to the use of NGS in diagnostic microbiology is the low ratio of pathogen DNA to human DNA in most clinical specimens. In this study, we aimed to develop a specimen-processing protocol to remove human DNA and enrich specimens for bacterial and viral DNA for shotgun metagenomic sequencing. Cerebrospinal fluid (CSF) and nasopharyngeal aspirate (NPA) specimens, spiked with control bacterial and viral pathogens, were processed using either a commercially available kit (MolYsis) or various detergents followed by DNase prior to the extraction of DNA. Relative quantities of human DNA and pathogen DNA were determined by real-time PCR. The MolYsis kit did not improve the pathogen-to-human DNA ratio, but significant reductions (>95%;P< 0.001) in human DNA with minimal effect on pathogen DNA were achieved in samples that were treated with 0.025% saponin, a nonionic surfactant. Specimen preprocessing significantly decreased NGS reads mapped to the human genome (P< 0.05) and improved the sensitivity of pathogen detection (P< 0.01), with a 20- to 650-fold increase in the ratio of microbial reads to human reads. Preprocessing also permitted the detection of pathogens that were undetectable in the unprocessed samples. Our results demonstrate a simple method for the reduction of background human DNA for metagenomic detection for a broad range of pathogens in clinical samples.
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http://dx.doi.org/10.1128/JCM.03050-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4809942PMC
April 2016

Total proteome analysis identifies migration defects as a major pathogenetic factor in immunoglobulin heavy chain variable region (IGHV)-unmutated chronic lymphocytic leukemia.

Mol Cell Proteomics 2015 Apr 2;14(4):933-45. Epub 2015 Feb 2.

From the ‡Department of Molecular and Clinical Cancer Medicine, ¶Royal Liverpool and Broadgreen University Hospitals NHS Trust, Liverpool L7 8XP, UK

The mutational status of the immunoglobulin heavy chain variable region defines two clinically distinct forms of chronic lymphocytic leukemia (CLL) known as mutated (M-CLL) and unmutated (UM-CLL). To elucidate the molecular mechanisms underlying the adverse clinical outcome associated with UM-CLL, total proteomes from nine UM-CLL and nine M-CLL samples were analyzed by isobaric tags for relative and absolute quantification (iTRAQ)-based mass spectrometry. Based on the expression of 3521 identified proteins, principal component analysis separated CLL samples into two groups corresponding to immunoglobulin heavy chain variable region mutational status. Computational analysis showed that 43 cell migration/adhesion pathways were significantly enriched by 39 differentially expressed proteins, 35 of which were expressed at significantly lower levels in UM-CLL samples. Furthermore, UM-CLL cells underexpressed proteins associated with cytoskeletal remodeling and overexpressed proteins associated with transcriptional and translational activity. Taken together, our findings indicate that UM-CLL cells are less migratory and more adhesive than M-CLL cells, resulting in their retention in lymph nodes, where they are exposed to proliferative stimuli. In keeping with this hypothesis, analysis of an extended cohort of 120 CLL patients revealed a strong and specific association between UM-CLL and lymphadenopathy. Our study illustrates the potential of total proteome analysis to elucidate pathogenetic mechanisms in cancer.
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http://dx.doi.org/10.1074/mcp.M114.044479DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4390271PMC
April 2015

ADAM17-dependent c-MET-STAT3 signaling mediates resistance to MEK inhibitors in KRAS mutant colorectal cancer.

Cell Rep 2014 Jun 12;7(6):1940-55. Epub 2014 Jun 12.

Centre for Cancer Research and Cell Biology, School of Medicine, Dentistry and Biomedical Science, Queen's University Belfast, 97 Lisburn Road, Belfast BT9 7AE, UK. Electronic address:

There are currently no approved targeted therapies for advanced KRAS mutant (KRASMT) colorectal cancer (CRC). Using a unique systems biology approach, we identified JAK1/2-dependent activation of STAT3 as the key mediator of resistance to MEK inhibitors in KRASMT CRC in vitro and in vivo. Further analyses identified acute increases in c-MET activity following treatment with MEK inhibitors in KRASMT CRC models, which was demonstrated to promote JAK1/2-STAT3-mediated resistance. Furthermore, activation of c-MET following MEK inhibition was found to be due to inhibition of the ERK-dependent metalloprotease ADAM17, which normally inhibits c-MET signaling by promoting shedding of its endogenous antagonist, soluble "decoy" MET. Most importantly, pharmacological blockade of this resistance pathway with either c-MET or JAK1/2 inhibitors synergistically increased MEK-inhibitor-induced apoptosis and growth inhibition in vitro and in vivo in KRASMT models, providing clear rationales for the clinical assessment of these combinations in KRASMT CRC patients.
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http://dx.doi.org/10.1016/j.celrep.2014.05.032DOI Listing
June 2014

Targeting treatment-resistant breast cancer stem cells with FKBPL and its peptide derivative, AD-01, via the CD44 pathway.

Clin Cancer Res 2013 Jul 5;19(14):3881-93. Epub 2013 Jun 5.

School of Pharmacy, Queen's University Belfast, Belfast, United Kingdom.

Purpose: FK506-binding protein like (FKBPL) and its peptide derivative, AD-01, have already shown tumor growth inhibition and CD44-dependent antiangiogenic activity. Here, we explore the ability of AD-01 to target CD44-positive breast cancer stem cells (BCSC).

Experimental Design: Mammosphere assays and flow cytometry were used to analyze the effect of FKBPL overexpression/knockdown and AD-01 treatment ± other anticancer agents on BCSCs using breast cancer cell lines (MCF-7/MDA-231/ZR-75), primary patient samples, and xenografts. Delays in tumor initiation were evaluated in vivo. The anti-stem cell mechanisms were determined using clonogenic assays, quantitative PCR (qPCR), and immunofluorescence.

Results: AD-01 treatment was highly effective at inhibiting the BCSC population by reducing mammosphere-forming efficiency and ESA(+)/CD44(+)/CD24(-) or aldehyde dehydrogenase (ALDH)(+) cell subpopulations in vitro and tumor initiation in vivo. The ability of AD-01 to inhibit the self-renewal capacity of BCSCs was confirmed; mammospheres were completely eradicated by the third generation. The mechanism seems to be due to AD-01-mediated BCSC differentiation shown by a significant decrease in the number of holoclones and an associated increase in meroclones/paraclones; the stem cell markers, Nanog, Oct4, and Sox2, were also significantly reduced. Furthermore, we showed additive inhibitory effects when AD-01 was combined with the Notch inhibitor, DAPT. AD-01 was also able to abrogate a chemo- and radiotherapy-induced enrichment in BCSCs. Finally, FKBPL knockdown led to an increase in Nanog/Oct4/Sox2 and an increase in BCSCs, highlighting a role for endogenous FKBPL in stem cell signaling.

Conclusions: AD-01 has dual antiangiogenic and anti-BCSC activity, which will be advantageous as this agent enters clinical trial.
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http://dx.doi.org/10.1158/1078-0432.CCR-13-0595DOI Listing
July 2013

Identification of galanin and its receptor GalR1 as novel determinants of resistance to chemotherapy and potential biomarkers in colorectal cancer.

Clin Cancer Res 2012 Oct 2;18(19):5412-26. Epub 2012 Aug 2.

Queen's University Belfast, Belfast, Northern Ireland.

Purpose: A major factor limiting the effective clinical management of colorectal cancer (CRC) is resistance to chemotherapy. Therefore, the identification of novel, therapeutically targetable mediators of resistance is vital.

Experimental Design: We used a CRC disease-focused microarray platform to transcriptionally profile chemotherapy-responsive and nonresponsive pretreatment metastatic CRC liver biopsies and in vitro samples, both sensitive and resistant to clinically relevant chemotherapeutic drugs (5-FU and oxaliplatin). Pathway and gene set enrichment analyses identified candidate genes within key pathways mediating drug resistance. Functional RNAi screening identified regulators of drug resistance.

Results: Mitogen-activated protein kinase signaling, focal adhesion, cell cycle, insulin signaling, and apoptosis were identified as key pathways involved in mediating drug resistance. The G-protein-coupled receptor galanin receptor 1 (GalR1) was identified as a novel regulator of drug resistance. Notably, silencing either GalR1 or its ligand galanin induced apoptosis in drug-sensitive and resistant cell lines and synergistically enhanced the effects of chemotherapy. Mechanistically, GalR1/galanin silencing resulted in downregulation of the endogenous caspase-8 inhibitor FLIP(L), resulting in induction of caspase-8-dependent apoptosis. Galanin mRNA was found to be overexpressed in colorectal tumors, and importantly, high galanin expression correlated with poor disease-free survival of patients with early-stage CRC.

Conclusion: This study shows the power of systems biology approaches to identify key pathways and genes that are functionally involved in mediating chemotherapy resistance. Moreover, we have identified a novel role for the GalR1/galanin receptor-ligand axis in chemoresistance, providing evidence to support its further evaluation as a potential therapeutic target and biomarker in CRC.
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http://dx.doi.org/10.1158/1078-0432.CCR-12-1780DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3463501PMC
October 2012

A systems biology approach identifies SART1 as a novel determinant of both 5-fluorouracil and SN38 drug resistance in colorectal cancer.

Mol Cancer Ther 2012 Jan 25;11(1):119-31. Epub 2011 Oct 25.

Centre for Cancer Research and Cell Biology, Queen's University Belfast, Belfast, Northern Ireland.

Chemotherapy response rates for advanced colorectal cancer remain disappointingly low, primarily because of drug resistance, so there is an urgent need to improve current treatment strategies. To identify novel determinants of resistance to the clinically relevant drugs 5-fluorouracil (5-FU) and SN38 (the active metabolite of irinotecan), transcriptional profiling experiments were carried out on pretreatment metastatic colorectal cancer biopsies and HCT116 parental and chemotherapy-resistant cell line models using a disease-specific DNA microarray. To enrich for potential chemoresistance-determining genes, an unsupervised bioinformatics approach was used, and 50 genes were selected and then functionally assessed using custom-designed short interfering RNA (siRNA) screens. In the primary siRNA screen, silencing of 21 genes sensitized HCT116 cells to either 5-FU or SN38 treatment. Three genes (RAPGEF2, PTRF, and SART1) were selected for further analysis in a panel of 5 colorectal cancer cell lines. Silencing SART1 sensitized all 5 cell lines to 5-FU treatment and 4/5 cell lines to SN38 treatment. However, silencing of RAPGEF2 or PTRF had no significant effect on 5-FU or SN38 sensitivity in the wider cell line panel. Further functional analysis of SART1 showed that its silencing induced apoptosis that was caspase-8 dependent. Furthermore, silencing of SART1 led to a downregulation of the caspase-8 inhibitor, c-FLIP, which we have previously shown is a key determinant of drug resistance in colorectal cancer. This study shows the power of systems biology approaches for identifying novel genes that regulate drug resistance and identifies SART1 as a previously unidentified regulator of c-FLIP and drug-induced activation of caspase-8.
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http://dx.doi.org/10.1158/1535-7163.MCT-11-0510DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3272421PMC
January 2012

Gene expression meta-analysis identifies VDAC1 as a predictor of poor outcome in early stage non-small cell lung cancer.

PLoS One 2011 Jan 31;6(1):e14635. Epub 2011 Jan 31.

Centre for Biomedical Informatics, Queen's University Belfast, Belfast, Northern Ireland, United Kingdom.

Background: The bioenergetic status of non-small cell lung cancer correlates with tumour aggressiveness. The voltage dependent anion channel type 1 (VDAC1) is a component of the mitochondrial permeability transition pore, regulates mitochondrial ATP/ADP exchange suggesting that its over-expression could be associated with energy dependent processes including increased proliferation and invasiveness. To test this hypothesis, we conducted an in vivo gene-expression meta-analysis of surgically resected non-small cell lung cancer (NSCLC) using 602 individual expression profiles, to examine the impact of VDAC1 on survival.

Methodology/principal Findings: High VDAC1 expression was associated with shorter overall survival with hazard ratio (HR) = 0.6639 (95% confidence interval (CI) 0.4528 to 0.9721), p = 0.035352 corresponding to 52 versus 101 months. VDAC1 predicted shorter time to recurrence and was shown to be an independent prognostic factor compared with histology, gender, age, nodal stage and tumour stage in a Cox multivariate analysis. Supervised analysis of all the datasets identified a 6-gene signature comprising HNRNPC, HSPA4, HSPA9, UBE2D2, CSNK1A1 and G3BP1 with overlapping functions involving regulation of protein turnover, RAS-RAF-MEK pathway and transcription. VDAC1 predicted survival in breast cancer and myeloma and an unsupervised analysis revealed enrichment of the VDAC1 signature in specific subsets.

Conclusions: In summary, gene expression analysis identifies VDAC1 gene expression as a predictor of poor outcome in NSCLC and other cancers and is associated with dysregulation of a conserved set of biological pathways, which may be causally associated with aggressive tumour behaviour.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0014635PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3031508PMC
January 2011

The colorectal cancer disease-specific transcriptome may facilitate the discovery of more biologically and clinically relevant information.

BMC Cancer 2010 Dec 20;10:687. Epub 2010 Dec 20.

Centre for Cancer Research and Cell Biology, Queen's University Belfast, Belfast, BT9 7BL, Northern Ireland.

Background: To date, there are no clinically reliable predictive markers of response to the current treatment regimens for advanced colorectal cancer. The aim of the current study was to compare and assess the power of transcriptional profiling using a generic microarray and a disease-specific transcriptome-based microarray. We also examined the biological and clinical relevance of the disease-specific transcriptome.

Methods: DNA microarray profiling was carried out on isogenic sensitive and 5-FU-resistant HCT116 colorectal cancer cell lines using the Affymetrix HG-U133 Plus2.0 array and the Almac Diagnostics Colorectal cancer disease specific Research tool. In addition, DNA microarray profiling was also carried out on pre-treatment metastatic colorectal cancer biopsies using the colorectal cancer disease specific Research tool. The two microarray platforms were compared based on detection of probesets and biological information.

Results: The results demonstrated that the disease-specific transcriptome-based microarray was able to out-perform the generic genomic-based microarray on a number of levels including detection of transcripts and pathway analysis. In addition, the disease-specific microarray contains a high percentage of antisense transcripts and further analysis demonstrated that a number of these exist in sense:antisense pairs. Comparison between cell line models and metastatic CRC patient biopsies further demonstrated that a number of the identified sense:antisense pairs were also detected in CRC patient biopsies, suggesting potential clinical relevance.

Conclusions: Analysis from our in vitro and clinical experiments has demonstrated that many transcripts exist in sense:antisense pairs including IGF2BP2, which may have a direct regulatory function in the context of colorectal cancer. While the functional relevance of the antisense transcripts has been established by many studies, their functional role is currently unclear; however, the numbers that have been detected by the disease-specific microarray would suggest that they may be important regulatory transcripts. This study has demonstrated the power of a disease-specific transcriptome-based approach and highlighted the potential novel biologically and clinically relevant information that is gained when using such a methodology.
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http://dx.doi.org/10.1186/1471-2407-10-687DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3018462PMC
December 2010

FKBPL regulates estrogen receptor signaling and determines response to endocrine therapy.

Cancer Res 2010 Feb 26;70(3):1090-100. Epub 2010 Jan 26.

School of Pharmacy, McClay Research Centre and Centre for Cancer Research and Cell Biology, Queen's University, Belfast, Northern Ireland BT9 7BL, United Kingdom.

The HSP90 chaperone and immunophilin FKBPL is an estrogen-responsive gene that interacts with estogen receptor alpha (ERalpha) and regulates its levels. In this study, we explored the effects of FKBPL on breast cancer proliferation. Breast cancer cells stably overexpressing FKBPL became dependent on estrogen for their growth and were dramatically more sensitive to the antiestrogens tamoxifen and fulvestrant, whereas FKBPL knockdown reverses this phenotype. FKBPL knockdown also decreased the levels of the cell cycle inhibitor p21WAF1 and increased ERalpha phosphorylation on Ser(118) in response to 17beta-estradiol and tamoxifen. In support of the likelihood that these effects explained FKBPL-mediated cell growth inhibition and sensitivity to endocrine therapies, FKBPL expression was correlated with increased overall survival and distant metastasis-free survival in breast cancer patients. Our findings suggest that FKBPL may have prognostic value based on its impact on tumor proliferative capacity and sensitivity to endocrine therapies, which improve outcome.
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http://dx.doi.org/10.1158/0008-5472.CAN-09-2515DOI Listing
February 2010

Clinical determinants of response to irinotecan-based therapy derived from cell line models.

Clin Cancer Res 2008 Oct;14(20):6647-55

Centre for Cancer Research and Cell Biology, Queen's University Belfast, Belfast, Northern Ireland.

Purpose: In an attempt to identify genes that are involved in resistance to SN38, the active metabolite of irinotecan (also known as CPT-11), we carried out DNA microarray profiling of matched HCT116 human colon cancer parental cell lines and SN38-resistant cell lines following treatment with SN38 over time.

Experimental Design: Data analysis identified a list of genes that were acutely altered in the parental cells following SN38 treatment as well as constitutively altered in the SN38-resistant cells.

Results: Independent validation of 20% of these genes by quantitative reverse transcription-PCR revealed a strong correlation with the microarray results: Pearson's correlation was 0.781 (r(2) = 0.61, P < 0.000001) for those genes that were acutely altered in the parental setting following SN38 treatment and 0.795 (r(2) = 0.63, P < 0.000002) for those genes that were constitutively altered in the SN38-resistant cells. We then assessed the ability of our in vitro-derived gene list to predict clinical response to 5-fluorouracil/irinotecan using pretreatment metastatic biopsies from responding and nonresponding colorectal cancer patients using both unsupervised and supervised approaches. When principal components analysis was used with our in vitro classifier gene list, a good separation between responding and nonresponding patients was obtained, with only one nonresponding and two responding patients separating with the incorrect groups. Supervised class prediction using support vector machines algorithm identified a 16-gene classifier with 75% overall accuracy, 81.8% sensitivity, and 66.6% specificity.

Conclusions: These results suggest that in vitro-derived gene lists can be used to predict clinical response to chemotherapy in colorectal cancer.
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http://dx.doi.org/10.1158/1078-0432.CCR-08-0452DOI Listing
October 2008
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