Imperial College London

DrLeandroCastellano

Faculty of MedicineDepartment of Surgery & Cancer

Visiting Reader
 
 
 
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Contact

 

+44 (0)20 7594 2822l.castellano Website

 
 
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Location

 

ICTEM buildingHammersmith Campus

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Summary

 

Publications

Publication Type
Year
to

82 results found

Pardo O, Chrysostomou S, Roy R, Prischi F, Thamlikitkul L, Chapman KL, Mufti U, Peach R, Ding L, Hancock D, Moore C, Molina-Arcas M, Mauri F, Pinato DJ, Abrahams JM, Ottaviani S, Castellano L, Giamas G, Pascoe J, Moonamale D, Pirrie S, Gaunt C, Billingham L, Steven NM, Cullen M, Hrouda D, Winkler M, Post J, Cohen P, Salpeter SJ, Bar V, Zundelevich A, Golan S, Leibovici D, Lara R, Klug DR, Yaliraki SN, Barahona M, Wang Y, Downward J, Skehel JM, Ali MMU, Seckl MJet al., 2021, Repurposed floxacins targeting RSK4 prevent chemoresistance and metastasis in lung and bladder cancer, Science Translational Medicine, Vol: 13, ISSN: 1946-6234

Lung and bladder cancers are mostly incurable because of the early development of drug resistance and metastatic dissemination. Hence, improved therapies that tackle these two processes are urgently needed to improve clinical outcome. We have identified RSK4 as a promoter of drug resistance and metastasis in lung and bladder cancer cells. Silencing this kinase, through either RNA interference or CRISPR, sensitized tumor cells to chemotherapy and hindered metastasis in vitro and in vivo in a tail vein injection model. Drug screening revealed several floxacin antibiotics as potent RSK4 activation inhibitors, and trovafloxacin reproduced all effects of RSK4 silencing in vitro and in/ex vivo using lung cancer xenograft and genetically engineered mouse models and bladder tumor explants. Through x-ray structure determination and Markov transient and Deuterium exchange analyses, we identified the allosteric binding site and revealed how this compound blocks RSK4 kinase activation through binding to an allosteric site and mimicking a kinase autoinhibitory mechanism involving the RSK4’s hydrophobic motif. Last, we show that patients undergoing chemotherapy and adhering to prophylactic levofloxacin in the large placebo-controlled randomized phase 3 SIGNIFICANT trial had significantly increased (P = 0.048) long-term overall survival times. Hence, we suggest that RSK4 inhibition may represent an effective therapeutic strategy for treating lung and bladder cancer.

Journal article

Malczewska A, Frampton AE, Mato Prado M, Ameri S, Dabrowska AF, Zagorac S, Clift AK, Kos-Kudła B, Faiz O, Stebbing J, Castellano L, Frilling Aet al., 2021, Circulating microRNAs in small-bowel neuroendocrine tumors: a potential tool for diagnosis and assessment of effectiveness of surgical resection, Annals of Surgery, Vol: 274, Pages: e1-e9, ISSN: 0003-4932

OBJECTIVE: To discover serum-based microRNA (miRNA) biomarkers for small-bowel neuroendocrine tumors (SBNET) to help guide clinical decisions. BACKGROUND: MiRNAs are small noncoding RNA molecules implicated in the initiation and progression of many cancers. MiRNAs are remarkably stable in bodily fluids, and can potentially be translated into clinically useful biomarkers. Novel biomarkers are needed in SBNET to determine disease aggressiveness, select patients for treatment, detect early recurrence, and monitor response. METHODS: This study was performed in 3 stages (discovery, validation, and a prospective, longitudinal assessment). Discovery comprised of global profiling of 376 miRNA in sera from SBNET patients (n = 11) versus healthy controls (HCs; n = 3). Up-regulated miRNAs were subsequently validated in additional SBNET (n = 33) and HC sera (n = 14); and then longitudinally after SBNET resection (n = 12), with serial serum sampling (preoperatively day 0; postoperatively at 1 week, 1 month, and 12 months). RESULTS: Four serum miRNAs (miR-125b-5p, -362-5p, -425-5p and -500a-5p) were significantly up-regulated in SBNET (P < 0.05; fold-change >2) based on multiple normalization strategies, and were validated by RT-qPCR. This combination was able to differentiate SBNET from HC with an area under the curve of 0.951. Longitudinal assessment revealed that miR-125b-5p returned towards HC levels at 1 month postoperatively in patients without disease, whereas remaining up-regulated in those with residual disease (RSD). This was also true at 12 months postoperatively. In addition, miR-362-5p appeared up-regulated at 12 months in RSD and recurrent disease (RCD). CONCLUSIONS: Our study represents the largest global profiling of serum miRNAs in SBNET patients, and the first to evaluate ongoing serum miRNA expression changes after surgical resection. Serum miR-125b-5p and miR-362-5p have potential to be used to detect RSD/RCD.

Journal article

Zagorac S, de Giorgio A, Dabrowska A, Kalisz M, Casas-Vila N, Cathcart P, Yiu A, Ottaviani S, Degani N, Lombardo Y, Tweedie A, Nissan T, Vance KW, Ulitsky I, Stebbing J, Castellano Let al., 2021, SCIRT lncRNA restrains tumorigenesis by opposing transcriptional programs of tumor-initiating cells., Cancer Research, Vol: 81, Pages: 580-593, ISSN: 0008-5472

In many tumors, cells transition reversibly between slow-proliferating tumor-initiating cells (TIC) and their differentiated, faster-growing progeny. Yet how transcriptional regulation of cell cycle and self-renewal genes is orchestrated during these conversions remains unclear. In this study, we show that as breast TIC form, a decrease in cell-cycle and increase in self-renewal gene expression is coregulated by SOX2 and EZH2, which colocalize at CpG islands. This pattern was negatively controlled by a novel long non-coding RNA (lncRNA) that we name SCIRT, which was markedly upregulated in tumorspheres but colocalized with and counteracted EZH2 and SOX2 during cell cycle and self-renewal regulation to restrain tumorigenesis. SCIRT specifically interacted with EZH2 to increase EZH2 affinity to FOXM1 without binding the latter. In this manner, SCIRT induced transcription at cell cycle gene promoters by recruiting FOXM1 through EZH2 to antagonize EZH2-mediated effects at target genes. Conversely, on stemness genes, FOXM1 was absent and SCIRT antagonized EZH2 and SOX2 activity, balancing towards repression. These data suggest that the interaction of a lncRNA with EZH2 can alter the affinity of EZH2 for its protein binding partners to regulate cancer cell state transitions.

Journal article

Ottaviani S, Stebbing J, Frampton AE, Zagorac S, Krell J, de Giorgio A, Trabulo SM, Nguyen VTM, Magnani L, Feng H, Giovannetti E, Funel N, Gress TM, Jiao LR, Lombardo Y, Lemoine NR, Heeschen C, Castellano Let al., 2019, Author Correction: TGF-beta induces miR-100 and miR-125b but blocks let-7a through LIN28B controlling PDAC progression, Nature Communications, Vol: 10, ISSN: 2041-1723

Journal article

Nguyen VTM, Barozzi I, Faronato M, Lombardo Y, Steel JH, Patel N, Darbre P, Castellano L, Gyorffy B, Woodley L, Rodriguez-Meira A, Patten DK, Vircillo V, Periyasamy M, Ali S, Frige G, Minucci S, Coombes RC, Magnani Let al., 2019, Author Correction: Differential epigenetic reprogramming in response to specific endocrine therapies promotes cholesterol biosynthesis and cellular invasion, Nature Communications, Vol: 10, ISSN: 2041-1723

Journal article

Chrysostomou S, Roy R, Prischi F, Chapman K, Mufti U, Mauri F, Bellezza G, Abrahams J, Ottaviani S, Castellano L, Giamas G, Hrouda D, Winkler M, Klug D, Yaliraki S, Barahona M, Wang Y, Ali M, Seckl M, Pardo Oet al., 2019, Targeting RSK4 prevents both chemoresistance and metastasis in lung cancer, AACR Annual Meeting on Bioinformatics, Convergence Science, and Systems Biology, Publisher: AMER ASSOC CANCER RESEARCH, ISSN: 0008-5472

Conference paper

Gall TMH, Gerrard G, Frampton AE, Castellano L, Ahmad R, Habib N, Spalding D, Pai M, Foroni L, Jiao LRet al., 2019, Can we predict long-term survival in resectable pancreatic ductal adenocarcinoma?, Oncotarget, Vol: 10, Pages: 696-706, ISSN: 1949-2553

Objective: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive tumour associated with poor 5-year survival. We aimed to determine factors which differentiate short and long-term survivors and identify a prognostic biomarker. Methods: Over a ten-year period, patients with resected PDAC who developed disease recurrence within 12 months (Group I) and those who had no disease recurrence for 24 months (Group II) were identified. Clinicopathological data was analysed. Ion Torrent high-throughput sequencing on DNA extracted from FFPE tumour samples was used to identify mutations. Additionally, peripheral blood samples were analysed for variants in cell-free DNA, circulating tumour cells (CTCs), and microRNAs. Results: Multivariable analysis of clinicopathological factors showed that a positive medial resection margin was significantly associated with short disease-free survival (p = 0.007). Group I patients (n = 21) had a higher frequency of the KRAS mutant mean variant allele (16.93% ± 11.04) compared to those in Group II (n = 13; 7.55% ± 5.76, p = 0.0078). Group I patients also trended towards having a KRAS c.35G>A p.Gly12Asp mutation in addition to variants in other genes, such as TP53, CDKN2A, and SMAD4. Mutational status of cell-free DNA, and number of CTCs, was not found to be useful in this study. A circulating miRNA (hsa-miR-548ah-5p) was found to be significantly differentially expressed. Conclusions: Medial resection margin status and the frequency of KRAS mutation in the tumour tissue are independent prognostic indicators for resectable PDAC. Circulating miRNA hsa-miR-548ah-5p has the potential to be used as a prognostic biomarker.

Journal article

Ottaviani S, Castellano L, 2018, microRNAs: novel regulators of the TGF- pathway in pancreatic ductal adenocarcinoma, Molecular & Cellular Oncology, Vol: 5, Pages: 1-3, ISSN: 2372-3556

We identified that transforming growth factor-β (TGF-β) induces long non-coding RNA (lncRNA) MIR100HG along with its host microRNAs (miRNAs) miR-100 and miR-125b, to regulate its response in pancreatic ductal adenocarcinoma (PDAC). Importantly let-7a, despite originating from MIR100HG, remains unchanged because post-transcriptionally repressed by lin-28 homolog B (LIN28B). A novel method for global miRNA-target discovery identified that miR-100/125b regulates crucial PDAC pathways.

Journal article

Blighe K, DeDionisio L, Christie KA, Chawes B, Shareef S, Kakouli-Duarte T, Chao-Shern C, Harding V, Kelly RS, Castellano L, Stebbing J, Lasky-Su JA, Nesbit MA, Moore CBTet al., 2018, Gene editing in the context of an increasingly complex genome, BMC GENOMICS, Vol: 19, ISSN: 1471-2164

Journal article

Begum S, Yiu A, Stebbing J, Castellano Let al., 2018, Novel tumour suppressive protein encoded by circular RNA, circ-SHPRH, in glioblastomas, ONCOGENE, Vol: 37, Pages: 4055-4057, ISSN: 0950-9232

Journal article

Ottaviani S, Stebbing J, Frampton AE, Zagorac S, Krell J, de Giorgio A, Trabulo SM, Nguyen VTM, Magnani L, Feng H, Giovannetti E, Funel N, Gress TM, Jiao LR, Lombardo Y, Lemoine NR, Heeschen C, Castellano Let al., 2018, TGF-beta induces miR-100 and miR-125b but blocks let-7a through LIN28B controlling PDAC progression, Nature Communications, Vol: 9, ISSN: 2041-1723

TGF-β/Activin induces epithelial-to-mesenchymal transition and stemness in pancreatic ductal adenocarcinoma (PDAC). However, the microRNAs (miRNAs) regulated during this response have remained yet undetermined. Here, we show that TGF-β transcriptionally induces MIR100HG lncRNA, containing miR-100, miR-125b and let-7a in its intron, via SMAD2/3. Interestingly, we find that although the pro-tumourigenic miR-100 and miR-125b accordingly increase, the amount of anti-tumourigenic let-7a is unchanged, as TGF-β also induces LIN28B inhibiting its maturation. Notably, we demonstrate that inactivation of miR-125b or miR-100 affects the TGF-β-mediated response indicating that these miRNAs are important TGF-β effectors. We integrate AGO2-RIP-seq with RNA-seq to identify the global regulation exerted by these miRNAs in PDAC cells. Transcripts targeted by miR-125b and miR-100 significantly overlap and mainly inhibit p53 and cell–cell junctions’ pathways. Together, we uncover that TGF-β induces an lncRNA, whose encoded miRNAs, miR-100, let-7a and miR-125b play opposing roles in controlling PDAC tumourigenesis.

Journal article

Frampton AE, Mato Prado M, Lopez Jimenez ME, Fajardo Puerta AB, Jawad ZR, Lawton P, Giovannetti E, Habib N, Castellano L, Stebbing J, Krell J, Jiao Let al., 2018, Glypican-1 is enriched in circulating-exosomes in pancreatic cancer and correlates with tumor burden, Oncotarget, Vol: 9, Pages: 19006-19013, ISSN: 1949-2553

Background: Glypican-1 (GPC1) is expressed in pancreatic ductal adenocarcinoma (PDAC) cells and adjacent stroma fibroblasts. Recently, GPC1 circulating exosomes (crExos) have been shown to be able to detect early stages of PDAC. This study investigated the usefulness of crExos GPC1 as a biomarker for PDAC.Methods: Plasma was obtained from patients with benign pancreatic disease (n = 16) and PDAC (n = 27) prior to pancreatectomy, and crExos were isolated by ultra-centrifugation. Protein was extracted from surgical specimens (adjacent normal pancreas, n = 13; and PDAC, n = 17). GPC1 levels were measured using enzyme-linked immunosorbent assay (ELISA). Results: There was no significant difference in GPC1 levels between normal pancreas and PDAC tissues. This was also true when comparing matched pairs. However, GPC1 levels were enriched in PDAC crExos (n = 11), compared to the source tumors (n = 11; 97 ± 54 vs. 20.9 ± 12.3 pg/mL; P < 0.001). In addition, PDACs with high GPC1 expression tended to have crExos with high GPC1 levels. Despite these findings, we were unable to distinguish PDAC from benign pancreatic disease using crExos GPC1 levels. Interestingly, we found that in matched pre and post-operative plasma samples there was a significant drop in crExos GPC1 levels after surgical resection for PDAC (n = 11 vs. 11; 97 ± 54 vs. 77.8 ± 32.4 pg/mL; P = 0.0428). Furthermore, we found that patients with high crExos GPC1 levels have significantly larger PDACs (>4 cm; P = 0.012). Conclusions: High GPC1 crExos may be able to determine PDAC tumor size and disease burden. However, further efforts are needed to elucidate its role as a diagnostic and/or prognostic biomarker using larger cohorts of PDAC patients.

Journal article

Macdougall CE, Wood EG, Loschko J, Scagliotti V, Cassidy FC, Robinson ME, Feldhahn N, Castellano L, Voisin M-B, Marelli-Berg F, Gaston-Massuet C, Charalambous M, Longhi MPet al., 2018, Visceral adipose tissue immune homeostasis Is regulated by the crosstalk between adipocytes and dendritic cell subsets, Cell Metabolism, Vol: 27, Pages: 588-601.e4, ISSN: 1550-4131

Visceral adipose tissue (VAT) has multiple roles in orchestrating whole-body energy homeostasis. In addition, VAT is now considered an immune site harboring an array of innate and adaptive immune cells with a direct role in immune surveillance and host defense. We report that conventional dendritic cells (cDCs) in VAT acquire a tolerogenic phenotype through upregulation of pathways involved in adipocyte differentiation. While activation of the Wnt/β-catenin pathway in cDC1 DCs induces IL-10 production, upregulation of the PPARγ pathway in cDC2 DCs directly suppresses their activation. Combined, they promote an anti-inflammatory milieu in vivo delaying the onset of obesity-induced chronic inflammation and insulin resistance. Under long-term over-nutrition, changes in adipocyte biology curtail β-catenin and PPARγ activation, contributing to VAT inflammation.

Journal article

Frampton AE, Funel N, Giovannetti E, Ahmad R, Castellano L, Knoesel T, Jiao LR, Stebbing J, Krell Jet al., 2017, y Dicing and slicing pancreatic cancer, 20th Annual Scientific Meeting of the Association-of-Upper-Gastrointestinal-Surgeons-of-Great-Britain-and-Ireland (AUGIS), Publisher: WILEY, Pages: 13-13, ISSN: 0007-1323

Conference paper

Frilling A, Kaemmerer D, Kidd MS, Clift AK, Stebbing J, Baum RP, Castellano L, Wasan HS, Modlin IMet al., 2017, Surgery in combination with peptide receptor radionuclide therapy is effective in metastatic neuroendocrine tumors and is definable by blood gene transcript analysis., 53rd Annual Clinical Science Meeting of the American-Society-of-Clinical-Oncology (ASCO) / Symposium on Old Targets, New Drugs - Her2 and MET, Publisher: AMER SOC CLINICAL ONCOLOGY, ISSN: 0732-183X

Conference paper

Boulianne B, Robinson ME, May PC, Castellano L, Blighe K, Thomas J, Reid A, Muschen M, Apperley JF, Stebbing J, Feldhahn Net al., 2017, Lineage-specific genes are prominent DNA damage hotspots during leukemic transformation of B-cell precursors, Cell Reports, Vol: 18, Pages: 1687-1698, ISSN: 2211-1247

In human leukemia, lineage-specific genes represent predominant targets of deletion, with lymphoid-specific genes frequently affected in lymphoid leukemia and myeloid-specific genes in myeloid leukemia. To investigate the basis of lineage-specific alterations, we analyzed global DNA damage in primary B-cell precursors expressing leukemia-inducing oncogenes by ChIP-Seq. We identified >1000 sensitive regions, of which B-lineage-specific genes constitute the most prominent targets. Identified hotspots at B-lineage genes relate to DNA-DSBs, affect genes that harbor genomic lesions in human leukemia, and associate with ectopic deletionin successfully transformed cells. We further show that mostidentified regions overlap with gene bodies of highly expressed genes, and that induction of a myeloidlineage phenotype in transformed B-cell precursors promotes de novoDNA damage atmyeloid loci. Hence, we demonstrate thatlineage-specific transcriptionpredisposeslineage-specificgenes in transformed B-cell precursorsto DNA damage, whichis likely to promote the frequent alteration oflineage-specific genes in human leukemia.

Journal article

Castellano L, Dabrowska A, Pellegrino L, Ottaviani S, Cathcart P, Frampton A, Krell J, Stebbing Jet al., 2017, Sustained expression of miR-26a promotes chromosomal instability and tumorigenesis through regulation of CHFR, Nucleic Acids Research, Vol: 45, Pages: 4401-4412, ISSN: 1362-4962

MicroRNA 26a (miR-26a) reduces cell viability in several cancers, indicating that miR-26a could be used as a therapeutic option in patients. We demonstrate that miR-26a not only inhibits G1-S cell cycle transition and promotes apoptosis, as previously described, but also regulates multiple cell cycle checkpoints. We show that sustained miR-26a over-expression in both breast cancer (BC) cell lines and mouse embryonic fibroblasts (MEFs) induces oversized cells containing either a single-large nucleus or two nuclei, indicating defects in mitosis and cytokinesis. Additionally, we demonstrate that miR-26a induces aneuploidy and centrosome defects and enhances tumorigenesis. Mechanistically, it acts by targeting G1-S transition genes as well as genes involved in mitosis and cytokinesis such as CHFR, LARP1 and YWHAE. Importantly, we show that only the re-expression of CHFR in miR-26a over-expressing cells partially rescues normal mitosis and impairs the tumorigenesis exerted by miR-26a, indicating that CHFR represents an important miR-26a target in the regulation of such phenotypes. We propose that miR-26a delivery might not be a viable therapeutic strategy due to the potential deleterious oncogenic activity of this miRNA.

Journal article

Prado MM, Frampton AE, Giovannetti E, Stebbing J, Castellano L, Krell Jet al., 2016, Investigating miRNA-mRNA regulatory networks using crosslinking immunoprecipitation methods for biomarker and target discovery in cancer, EXPERT REVIEW OF MOLECULAR DIAGNOSTICS, Vol: 16, Pages: 1155-1162, ISSN: 1473-7159

Journal article

Harrod A, Fulton J, Nguyen VTM, Periyasamy M, Ramos Garcia L, Lai C-F, Metodieva G, de Giorgio A, Williams RL, Santos DB, Jimenez Gomez P, Lin M-L, Metodiev MV, Stebbing J, Castellano L, Magnani L, Coombes RC, Buluwela L, Ali Set al., 2016, Genomic modelling of the ESR1 Y537S mutation for evaluating function and new therapeutic approaches for metastatic breast cancer, Oncogene, Vol: 36, Pages: 2286-2296, ISSN: 1476-5594

Drugs that inhibit estrogen receptor-α (ER) activity have been highlysuccessful in treating and reducing breast cancer progression in ER-positivedisease. However, resistance to these therapies presents a major clinicalproblem. Recent genetic studies have shown that mutations in the ER geneare found in >20% of tumours that progress on endocrine therapies.Remarkably, the great majority of these mutations localise to just a few aminoacids within or near the critical helix 12 region of the ER hormone bindingdomain, where they are likely to be single allele mutations. Understandinghow these mutations impact on ER function is a prerequiste for identifyingmethods to treat breast cancer patients featuring such mutations. Towardsthis end, we used CRISPR-Cas9 genome editing to make a single alleleknockin of the most commonly mutated amino acid residue, tyrosine 537, inthe estrogen-responsive MCF7 breast cancer cell line. Genomic analysesusing RNA-seq and ER ChIP-seq demonstrated that the Y537S mutationpromotes constitutive ER activity globally, resulting in estrogen-independentgrowth. MCF7-Y537S cells were resistant to the anti-estrogen tamoxifen andfulvestrant. Further, we show that the basal transcription factor TFIIH isconstitutively recruited by ER-Y537S, resulting in ligand-independentphosphorylation of Serine 118 (Ser118) by the TFIIH kinase, CDK7. TheCDK7 inhibitor, THZ1 prevented Ser118 phosphorylation and inhibited growthof MCF7-Y537S cells. These studies confirm the functional importance of ERmutations in endocrine resistance, demonstrate the utility of knockinmutational models for investigating alternative therapeutic approaches andhighlight CDK7 inhibition as a potential therapy for endocrine resistant breastcancer mediated by ER mutations.

Journal article

Miller HC, Frampton AE, Malczewska A, Ottaviani S, Stronach EA, Flora R, Kaemmerer D, Schwach G, Pfragner R, Faiz O, Kos-Kudła B, Hanna GB, Stebbing J, Castellano L, Frilling Aet al., 2016, MicroRNAs associated with small bowel neuroendocrine tumours and their metastases, Endocrine-Related Cancer, Vol: 23, Pages: 711-726, ISSN: 1479-6821

Novel molecular analytes are needed in small bowel neuroendocrine tumours (SBNETs) to better determine disease aggressiveness and predict treatment response. In this study, we aimed to profile the global miRNome of SBNETs, and identify microRNAs (miRNAs) involved in tumour progression for use as potential biomarkers. Two independent miRNA profiling experiments were performed (n=90), including primary SBNETs (n=28), adjacent normal small bowel (NSB; n=14), matched lymph node (LN) metastases (n=24), normal LNs (n=7), normal liver (n=2) and liver metastases (n=15). We then evaluated potentially targeted genes by performing integrated computational analyses. We discovered 39 miRNAs significantly deregulated in SBNETs compared with adjacent NSB. The most upregulated (miR-204-5p, miR-7-5p and miR-375) were confirmed by qRT-PCR. Two miRNAs (miR-1 and miR-143-3p) were significantly downregulated in LN and liver metastases compared with primary tumours. Furthermore, we identified upregulated gene targets for miR-1 and miR-143-3p in an existing SBNET dataset, which could contribute to disease progression, and show that these miRNAs directly regulate FOSB and NUAK2 oncogenes. Our study represents the largest global miRNA profiling of SBNETs using matched primary tumour and metastatic samples. We revealed novel miRNAs deregulated during SBNET disease progression, and important miRNA–mRNA interactions. These miRNAs have the potential to act as biomarkers for patient stratification and may also be able to guide treatment decisions. Further experiments to define molecular mechanisms and validate these miRNAs in larger tissue cohorts and in biofluids are now warranted.

Journal article

Stebbing J, Frampton AE, Miller HC, Malczewska A, Ottaviani S, Stronach EA, Flora R, Kaemmerer D, Schwach G, Pfragner R, Faiz O, Hanna G, Castellano L, Frilling Aet al., 2016, MicroRNAs associated with small bowel neuroendocrine tumors and their metastases., Annual Meeting of the American-Society-of-Clinical-Oncology (ASCO), Publisher: AMER SOC CLINICAL ONCOLOGY, ISSN: 0732-183X

Conference paper

Frampton AE, Krell J, Mato Prado M, Gall T, Abbassi-Ghadi N, Del Vecchio Blanco G, Funel N, Giovannetti E, Castellano L, Basyouny M, Habib N, Kaltsidis H, Vlavianos P, Stebbing J, Jiao Let al., 2016, Prospective validation of microRNA signatures for detecting pancreatic malignant transformation in endoscopic-ultrasound guided fine-needle aspiration biopsies, Oncotarget, Vol: 7, ISSN: 1949-2553

Background: Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease. Novel biomarkers are required to aid treatment decisions and improve patient outcomes. MicroRNAs (miRNAs) are potentially ideal diagnostic biomarkers, as they are stable molecules, and tumour and tissue specific. Results: Logistic regression analysis revealed an endoscopic-ultrasound fine-needle aspiration (EUS-FNA) 2-miRNA classifier (miR-21 + miR-155) capable of distinguishing benign from malignant pancreatic lesions with a sensitivity of 81.5% and a specificity of 85.7% (AUC 0.930). Validation FNA cohorts confirmed both miRNAs were overexpressed in malignant disease, while circulating miRNAs performed poorly.Methods: Fifty-five patients with a suspicious pancreatic lesion on cross-sectional imaging were evaluated by EUS-FNA. At echo-endoscopy, the first part of the FNA was sent for cytological assessment and the second part was used for total RNA extraction. Candidate miRNAs were selected after careful review of the literature and expression was quantified by qRT-PCR. Validation was performed on an independent cohort of EUS-FNAs, as well as formalin-fixed paraffin embedded (FFPE) and plasma samples.Conclusions: We provide further evidence for using miRNAs as diagnostic biomarkers for pancreatic malignancy. We demonstrate the feasibility of using fresh EUS-FNAs to establish miRNA-based signatures unique to pancreatic malignant transformation and the potential to enhance risk stratification and selection for surgery.

Journal article

Krell J, Stebbing J, Carissimi C, Dabrowska AF, de Giorgio A, Frampton AE, Harding V, Fulci V, Macino G, Colombo T, Castellano Let al., 2016, TP53 regulates miRNA association with AGO2 to remodel the miRNA-mRNA interaction network, GENOME RESEARCH, Vol: 26, Pages: 331-341, ISSN: 1088-9051

Journal article

Pardo OE, Munro CE, Castellano L, Hu Y, Mauri F, Krell J, Lara R, Pinho FG, Pellegrino L, Pshezhetskiy D, Wang Y, Waxman J, Seckl MJ, Stebbing Jet al., 2016, miR-515-5p controls cancer cell migration through MARK4 regulation, EMBO Reports, Vol: 17, Pages: 570-584, ISSN: 1469-221X

Here we show that miR-515-5p inhibits cancer cell migration and metastasis. RNA-seqanalyses of both estrogen receptor-positive and negative breast cancer cells overexpressingmiR-515-5p reveals down-regulation of NRAS, FZD4, CDC42BPA, PIK3C2Band MARK4 mRNAs. We demonstrate that miR-515-5p inhibits MARK4 directly 3’UTRinteraction and that MARK4 knockdown mimics the effect of miR-515-5p on breast andlung cancer cell migration. MARK4 over-expression rescues the inhibitory effects of miR-515-5p, suggesting miR-515-5p mediates this process through MARK4 down-regulation.Furthermore, miR-515-5p expression is reduced in metastases compared to primarytumours derived from both in vivo xenografts and samples from patients with breastcancer. Conversely, miR-515-5p overexpression prevents tumour cell dissemination ina mouse metastatic model. Moreover, high miR-515-5p and low MARK4 expressioncorrelate with increased breast and lung cancer patients’ survival, respectively. Takentogether, these data demonstrate the importance of miR-515-5p/MARK4 regulation incell migration and metastasis across two common cancers.

Journal article

Jacob J, Favicchio R, Karimian N, Mehrabi M, Harding V, Castellano L, Stebbing J, Giamas Get al., 2015, LMTK3 escapes tumour suppressor miRNAs via sequestration of DDX5., Cancer Letters, Vol: 372, Pages: 137-146, ISSN: 1872-7980

Lemur tyrosine kinase-3 (LMTK3) plays an important role in cancer progression and is associated with breast, lung, gastric and colorectal cancer. MicroRNAs (miRNAs) are small endogenous non-coding RNAs that typically repress target genes at post-transcriptional level and have an important role in tumorigenesis. By performing a miRNA expression profile, we identified a subset of miRNAs modulated by LMTK3. We show that LMTK3 induces miR-34a, miR-196-a2 and miR-182 levels interacting with DEAD-box RNA helicase p68 (DDX5). LMTK3 binds via DDX5 to the pri-miRNA of these three mature miRNAs, thereby sequestrating them from further processing. Ectopic expression of miR-34a and miR-182 in LMTK3-overexpressing cell lines (MCF7-LMTK3 and MDA-MB-231-LMTK3) inhibits breast cancer proliferation, invasion and migration. Interestingly, miR-34a and miR-182 directly bind to the 3'UTR of LMTK3 mRNA and consequently inhibit both its stability and translation, acting as tumour suppressor-like miRNAs. In aggregate, we show that LMTK3 is involved in miRNA biogenesis through modulation of the Microprocessor complex, inducing miRNAs that target LMTK3 itself.

Journal article

Krell J, Stebbing J, Carissimi C, Dabrowska AF, de Giorgio A, Frampton AE, Harding V, Fulci V, Macino G, Colombo T, Castellano Let al., 2015, TP53 regulates miRNA association with AGO2 to remodel the miRNA-mRNA interaction network., Genome Research, ISSN: 1549-5469

DNA damage activates TP53-regulated surveillance mechanisms that are crucial in suppressing tumorigenesis. TP53 orchestrates these responses directly by transcriptionally modulating genes, including microRNAs (miRNAs), and by regulating miRNA biogenesis through interacting with the DROSHA complex. However, whether the association between miRNAs and AGO2 is regulated following DNA damage is not yet known. Here we show that, following DNA damage, TP53 interacts with AGO2 to induce or reduce AGO2's association of a subset of miRNAs, including multiple let-7 family members. Furthermore, we show that specific mutations in TP53 decrease rather than increase the association of let-7 family miRNAs, reducing their activity without preventing TP53 from interacting with AGO2. This is consistent with the oncogenic properties of these mutants. Using AGO2 RIP-seq and PAR-CLIP-seq we show that the DNA-damage-induced increase in binding of let-7 family members to the RISC complex is functional. We unambiguously determine the global miRNA-mRNA interaction networks involved in the DNA damage response, validating them through the identification of miRNA-target chimeras formed by endogenous ligation reactions. We find that the target complementary region of the let-7 seed tends to have highly fixed positions and more variable ones. Additionally, we observe that miRNAs whose cellular abundance or differential association with AGO2 is regulated by TP53 are involved in an intricate network of regulatory feedback and feedforward circuits. TP53-mediated regulation of AGO2-miRNA interaction represents a new mechanism of miRNA regulation in carcinogenesis.

Journal article

Nguyen VTM, Barozzi I, Faronato M, Lombardo Y, Steel JH, Patel N, Darbre P, Castellano L, Gyorffy B, Woodley L, Meira A, Patten DL, Vircillo V, Periyasamy M, Ali S, Frige G, Minucci S, Coombes RC, Magnani Let al., 2015, Differential epigenetic reprogramming in response to specific endocrine therapies promotes cholesterol biosynthesis and cellular invasion, Nature Communications, Vol: 6, Pages: 1-15, ISSN: 2041-1723

Endocrine therapies target the activation of the oestrogen receptor alpha (ERa) via distinctmechanisms, but it is not clear whether breast cancer cells can adapt to treatment usingdrug-specific mechanisms. Here we demonstrate that resistance emerges via drug-specificepigenetic reprogramming. Resistant cells display a spectrum of phenotypical changes withinvasive phenotypes evolving in lines resistant to the aromatase inhibitor (AI). Orthogonalgenomics analysis of reprogrammed regulatory regions identifies individual drug-inducedepigenetic states involving large topologically associating domains (TADs) and the activationof super-enhancers. AI-resistant cells activate endogenous cholesterol biosynthesis (CB)through stable epigenetic activation in vitro and in vivo. Mechanistically, CB sparks theconstitutive activation of oestrogen receptors alpha (ERa) in AI-resistant cells, partly via thebiosynthesis of 27-hydroxycholesterol. By targeting CB using statins, ERa binding is reducedand cell invasion is prevented. Epigenomic-led stratification can predict resistance to AI in asubset of ERa-positive patients.

Journal article

Cathcart P, Lucchesi W, Ottaviani S, De Giorgio A, Krell J, Stebbing J, Castellano Let al., 2015, Noncoding RNAs and the control of signalling via nuclear receptor regulation in health and disease, BEST PRACTICE & RESEARCH CLINICAL ENDOCRINOLOGY & METABOLISM, Vol: 29, Pages: 529-543, ISSN: 1521-690X

Journal article

Frampton AE, Krell J, Gall TMH, Castellano L, Stebbing J, Jiao LRet al., 2015, miR-15b and miR-17 Are Tumor-derived Plasma MicroRNAs Dysregulated in Colorectal Neoplasia, Annals of Surgery, Vol: 262, Pages: e61-e61, ISSN: 0003-4932

Journal article

Frampton AE, Krell J, Jamieson NB, Gall TMH, Giovannetti E, Funel N, Prado MM, Krell D, Habib NA, Castellano L, Jiao LR, Stebbing Jet al., 2015, microRNAs with prognostic significance in pancreatic ductal adenocarcinoma: A meta-analysis, European Journal of Cancer, Vol: 51, Pages: 1389-1404, ISSN: 1879-0852

BackgroundReports have described the prognostic relevance of microRNAs (miRNAs) in patients treated for pancreatic ductal adenocarcinoma (PDAC). However, many of these include small numbers of patients. To increase statistical power and improve translation, we performed a systematic review and meta-analysis to determine a pooled conclusion. We examined the impact of miRNAs on overall survival (OS) and disease-free survival (DFS) in PDAC.MethodsEligible studies were identified and quality assessed using multiple search strategies (last search December 2014). Data were collected from studies correlating clinical outcomes with dysregulated tumoural or blood miRNAs. Studies were pooled, and combined hazard ratios (HRs) with 95% confidence intervals (CIs) were used to estimate strength of the associations.ResultsTwenty studies involving 1525 patients treated for PDAC were included. After correcting for publication bias, OS was significantly shortened in patients with high tumoural miR-21 (adjusted HR = 2.48; 1.96–3.14). This result persisted when only studies adjusting for adjuvant chemotherapy were combined (adjusted HR = 2.72; 1.91–3.89). High miR-21 also predicted reduced DFS (adjusted HR = 3.08; 1.78–5.33). Similarly, we found significant adjusted HRs for poor OS for high miR-155, high miR-203, and low miR-34a; and unadjusted HRs for high miR-222 and high miR-10b. The small number of studies, limited number of miRNAs and paucity of multivariate analyses are the limitations of our study.ConclusionsThis is the first rigorous pooled analysis assessing miRNAs as prognostic biomarkers in PDAC. Tumoural miR-21 overexpression emerged as an important predictor of poor prognosis after PDAC resection independent of other clinicopathologic factors, including adjuvant chemotherapy use.

Journal article

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