sumeth perera - Academia.edu (original) (raw)

Papers by sumeth perera

medRxiv (Cold Spring Harbor Laboratory), Jun 21, 2024

Oncogene, Oct 5, 2015

Tumour cells can use strategies that make them resistant to nutrient deprivation to outcompete th... more 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.

Journal of Oral Pathology & Medicine, Apr 18, 2007

BACKGROUND: Epidemiological data have shown an association of areca nut chewing with oral submuco... more BACKGROUND: Epidemiological data have shown an association of areca nut chewing with oral submucous fibrosis (OSF). Experimental evidence to confirm this has been limited. Fibrosis-promoting activity of areca nut was tested in an animal model. METHOD: Buccal mucosa of a group of 20 female BALB/ c strain mice, 10-12 weeks of age, was treated twice daily 6 days per week with topical application of aqueous areca nut extracts for 300-600 days. A control group (n ¼ 20) was treated with 50 mM NaCl. The influence of areca nut on the oral epithelium and connective tissue was recorded semiquantitatively by light microscopy. RESULTS: The areca nut-treated oral epithelium showed progressive changes in epithelial thickness leading to atrophy, increased cellularity of fibroblasts, fibrosis of connective tissue, focal infiltration of inflammatory cells and muscle atrophy. On killing after 600 days of treatment, the scores on cellularity, inflammation and muscle atrophy were significantly different to the control group (P ¼ 0.03). CONCLUSION: The study provides further evidence that areca nut contributes to the development of OSF in treated animals. The model has the potential to test synergism of areca nut with other carcinogens and any therapeutic interventions.

PLOS Genetics, Oct 11, 2016

Regulated secretion by glands and neurons involves release of signalling molecules and enzymes se... more Regulated secretion by glands and neurons involves release of signalling molecules and enzymes selectively concentrated in dense-core granules (DCGs). Although we understand how many secretagogues stimulate DCG release, how DCG biogenesis is then accelerated to replenish the DCG pool remains poorly characterised. Here we demonstrate that each prostate-like secondary cell (SC) in the paired adult Drosophila melanogaster male accessory glands contains approximately ten large DCGs, which are loaded with the Bone Morphogenetic Protein (BMP) ligand Decapentaplegic (Dpp). These DCGs can be marked in living tissue by a glycophosphatidylinositol (GPI) lipid-anchored form of GFP. In virgin males, BMP signalling is sporadically activated by constitutive DCG secretion. Upon mating, approximately four DCGs are typically released immediately, increasing BMP signalling, primarily via an autocrine mechanism. Using inducible knockdown specifically in adult SCs, we show that secretion requires the Soluble NSF Attachment Protein, SNAP24. Furthermore, mating-dependent BMP signalling not only promotes cell growth, but is also necessary to accelerate biogenesis of new DCGs, restoring DCG number within 24 h. Our analysis therefore reveals an autocrine BMP-mediated feedback mechanism for matching DCG release to replenishment as secretion rates fluctuate, and might explain why in other disease-relevant systems, like pancreatic β-cells, BMP signalling is also implicated in the control of secretion.

EMBO reports

Motile cilia on the cell surface generate movement and directional fluid flow that is crucial for... more Motile cilia on the cell surface generate movement and directional fluid flow that is crucial for various biological processes. Dysfunction of these cilia causes human diseases such as sinopulmonary disease and infertility. Here, we show that Ccdc108, a protein linked to male infertility, has an evolutionarily conserved requirement in motile multiciliation. Using Xenopus laevis embryos, Ccdc108 is shown to be required for the migration and docking of basal bodies to the apical membrane in epidermal multiciliated cells (MCCs). We demonstrate that Ccdc108 interacts with the IFT‐B complex, and the ciliation requirement for Ift74 overlaps with Ccdc108 in MCCs. Both Ccdc108 and IFT‐B proteins localize to migrating centrioles, basal bodies, and cilia in MCCs. Importantly, Ccdc108 governs the centriolar recruitment of IFT while IFT licenses the targeting of Ccdc108 to the cilium. Moreover, Ccdc108 is required for the centriolar recruitment of Drg1 and activated RhoA, factors that help establish the apical actin network in MCCs. Together, our studies indicate that Ccdc108 and IFT‐B complex components cooperate in multiciliogenesis.

A. Live image of Dpp-GFP-expressing SC stained with Lysotracker Red t... more A. Live image of Dpp-GFP-expressing SC stained with Lysotracker Red to identify acidic compartments. Note that Dpp-GFP localises to spherical structures (arrows) that are distinct from acidic compartments. B. Image of fixed SC, stained with anti-ANCE antibody (red) and DAPI (blue) after 24 h pulse of Dpp-GFP expression, reveals co-localisation of GFP fluorescence with ANCE-positive DCGs (arrows). C. Rab11-YFP-positive compartments in SCs from flies ubiquitously expressing this fusion protein under tubulin promoter control contain DCGs that stain positive for ANCE (red). D. Fixed SC expressing GFP-GPI and stained with anti-ANCE (red) and DAPI (blue), showing co-localisation of ANCE and GFP in DCGs (arrows). E. GFP-positive puncta (arrow) and filaments (arrowhead) are detected in the AG lumen when Dpp-GFP is expressed in SCs. F, G. SC-specific expression of Snap24 RNAi in adults significantly increases number of GFP-GPI-labelled DCGs in 6-day-old virgin males (F; using spi-GAL4 driver), and reduces nuclear size (G; using esgF/Ots driver). All images are from 3-day-old virgin males and individual SCs are outlined by dashed circles. Genotypes for images are: w; tub-GAL80ts; dsx-GAL4/UAS-Dpp-GFP (A, B, E); w; tub-rab11-YFP (C); w; spi-GAL4 tub-GAL80ts UAS-GFP-GPI/CyO (D). ***P<0.001, Mann-Whitney U test, n = 10. Scale bar for A-D is 10 μm and E is 20 μm.</p

A. Schematic of the paired male accessory glands (arrows), which pump... more A. Schematic of the paired male accessory glands (arrows), which pump their contents into the ejaculatory duct (arrowhead) during mating. Left inset shows the epithelial secretory monolayer containing secondary cells (SCs; green) and main cells (MCs), all of which are binucleate. Right inset is a section through the gland revealing the large lumen (asterisk). B. SC (circled) expressing a gene trap for the BMP type I receptor tkv, and stained with an antibody against the BMP type II receptor Wit. These receptors are present both on the plasma membrane (arrowhead) and co-localise in intracellular compartments (arrow). DAPI marks nuclei blue in SCs (asterisks) and surrounding non-expressing MCs. C-F. Accessory glands (AGs) from 6-day-old controls (C) and males expressing RNAis targeting tkv (D) or Mad (F) or expressing the BMP signalling antagonist Dad (E) in adult SCs under the control of esgF/Ots after temperature shift at eclosion. AGs were dissected, fixed and imaged by confocal microscopy. Glands were stained with DAPI (blue) to mark nuclei and an anti-Fas3 antibody (yellow) to mark cell boundaries. SCs express nuclear GFP, which is also present in the cytosol. Pairs of nuclei from binucleate SCs and MCs are indicated by green and red arrows respectively. G. Bar chart showing SC nuclear size relative to that of MC neighbours for glands expressing different transgenes in SCs under esgF/Ots control, normalized to the ratio for controls. Note that all manipulations that decrease BMP signalling significantly reduce SC nuclear size. Genotypes for images are: w; PBac[544.SVS-1]tkvCPTI002487 (B); w; esg-GAL4 tub-GAL80ts UAS-FLP; UAS-GFPnls actin>FRT>CD2>FRT>GAL4 combined with no other transgene (C); P[TRiP.JF01485]attP2 (III) (D); P[w+ UAS-Dad] (II) (E); P[TRiP.JF01263]attP2 (III) (F). ***P<0.001, Kruskal-Wallis test, n = 10. Scale bar for (B) is 10 μm, for all other images it is 20 μm.</p

Schematics of a single SC immediately before and after mating. (1) In virgin males, Dpp ... more Schematics of a single SC immediately before and after mating. (1) In virgin males, Dpp is trafficked to and stored in DCGs. Sporadic release of these DCGs activates BMP signalling, and sustains a basal level of growth, DCG biogenesis and exosome secretion. (2) During mating, about 4 mature DCGs are released (3), resulting in an increase in BMP signalling (4), primarily via an autocrine mechanism and probably in pulses. This stimulates growth, but also increases biosynthesis of new DCG compartments (5; solid arrow), ensuring that the total number of DCGs is fully replenished within 24 h. Dashed arrows highlight other parts of the secretory/endolysosomal system that might be affected by altered BMP signalling. Previous data (Corrigan et al., 2014) and data presented here suggest that long-term elevated BMP signalling enhances endolysosomal trafficking.</p

A, B. Single z-plane images of SCs from 6-day-old virgin males labell... more A, B. Single z-plane images of SCs from 6-day-old virgin males labelled with GFP-GPI, either co-expressing (B) or not expressing (A) dpp-RNAi. Reducing Dpp signalling decreases the number of labelled DCGs (arrow). C-E. 6-day-old males were shifted to 28.5°C to induce GFP-GPI expression for 16 hrs in virgins (C) or immediately after (D) or before (E) mating. Their AGs were dissected and imaged; each image is from a single z-plane. F. Graph showing number of GFP-GPI-positive DCGs in SCs from either 6-day-old virgin or mated control flies or flies in which BMP signalling is inhibited. There is no decrease in total DCG number in SCs expressing either dpp-RNAi or Dad after mating. G. Graph showing GFP-positive DCG number after a 16 h pulse of GFP-GPI in SCs from 6-day-old males, as in C-E above. Genotypes for images are: w; spi-GAL4 tub-GAL80ts UAS-GFP-GPI combined with no other transgene (A, and pulse-labelled in C, D, E) or P[TRiP.HMS00011]attP2 (III) (B). The w; spi-GAL4 tub-GAL80ts UAS-GFP-GPI line was used to generate data in F and G. ***P<0.001, Kruskal-Wallis test, n = 15. Scale bar for A-E, 10 μm.</p

Cancer Research

Genome wide association studies (GWAS) in 9,013 pancreatic cancer patients and 12,452 controls of... more Genome wide association studies (GWAS) in 9,013 pancreatic cancer patients and 12,452 controls of European ancestry have discovered over 20 risk loci in the human genome. Here, we fine mapped one such locus on chromosome 16q23.1 in the vicinity of two Chymotrypsinogen precursor genes, CTRB1 and CTRB2. We fine-mapped this locus to rs72802365 (P=2.51x10-17, OR=1.36, 95% CI=1.31-1.40) and identified colocalization (PP=87%) with aberrant exon 5→7 CTRB2 splicing in pancreatic tissues. Imputation of a 584 bp insertion/deletion variant overlapping exon 6 of CTRB2 into the GWAS datasets resulted in a highly significant association with pancreatic cancer risk (P=2.83x10-16, OR=1.36, 95% CI=1.31-1.42), indicating that it may underlie this signal. Exon skipping attributable to the deletion (risk) allele introduces a premature stop codon in exon 7 of CTRB2, yielding a truncated chymotrypsin B2 protein that lacks chymotrypsin activity, is poorly secreted, and accumulates intracellularly in the e...

A. Schematic representation of pulse-chase experiments shown in B-F, ... more A. Schematic representation of pulse-chase experiments shown in B-F, indicating the duration of GFP-GPI and Dad overexpression and timings of mating events. B-D. 6-day-old flies were shifted to 28.5°C for 24 h to allow expression of GFP-GPI in virgins (B) or in males mated 8 h after the start of the pulse (C). The number of GFP-GPI-labelled DCGs in SCs was reduced in virgin males co-expressing Dad (D). E. Graph shows a significant increase in the number of labelled DCGs if males are mated at 8h during a 24 h GFP-GPI pulse. The number of DCGs labelled in virgin and mated males is reduced if Dad is co-expressed. F. The increase in labelled compartments after mating is also reduced by Dad co-expression. The w; spi-GAL4 tub-GAL80ts UAS-GFP-GPI line was used to generate data in E and F. *P<0.05, ***P<0.001, Kruskal-Wallis test, n = 15. Scale bar in B-D, 10 μm.</p

A, B. SCs from 6-day-old esgF/Ots virgin males... more A, B. SCs from 6-day-old esgF/Ots virgin males (A) and from males immediately after mating (B) were fixed and stained with an anti-pMad antibody (red) and DAPI (blue), revealing that the proportion of SCs with detectable nuclear pMad is higher in mated animals. C, D. Immediately after mating, living SCs (D) have less GFP-GPI-labelled DCGs than virgins (C). Image shows a single z-plane of gland stained with Lysotracker Red; not all compartments are in the focal plane. Note that the largest MVBL in (C; arrowhead) contains GFP, probably because of fusion between a DCG compartment and the MVBL [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006366#pgen.1006366.ref017" target="_blank">17</a>]. E. Graph shows proportion of SCs with nuclear pMad in 6-day-old virgin, and mated males (dissected 8 min into mating [Mid] and immediately after mating), and mated males expressing dpp-RNAi and Dad in SCs from eclosion onwards using the w; esg-GAL4 tub-GAL80ts UAS-FLP; UAS-GFPnls actin>FRT>CD2>FRT>GAL4 driver. F. Graph shows number of GFP-GPI-positive DCG compartments in 6-day-old virgin and mated males (using the w; spi-GAL4 tub-GAL80ts UAS-GFP-GPI driver line; Double is twice mated in 2 h), and at different times after single mating in control SCs. Compartments were also counted in SCs expressing Snap24 RNAi post-eclosion in virgins and immediately after mating. Labelled compartments were counted using a complete z-series for each cell. Genotypes for images are: w; esg-GAL4 tub-GAL80ts UAS-FLP/+; UAS-GFPnls actin>FRT>CD2>FRT>GAL4/+ (A, B); w; spi-GAL4 tub-GAL80ts UAS-GFP-GPI/CyO (C, D).***P<0.001, Kruskal-Wallis test, n>15. Scale bar for A-B is 20 μm and C-D is 10 μm.</p

A. Expression of dpp-RNAi during the first six day... more A. Expression of dpp-RNAi during the first six days of adulthood using the esgF/Ots driver reduces the size of SCs and their nuclei (green arrows) relative to MCs (red arrows). B. Relative SC:MC nuclear size for SCs expressing RNAis targeting dpp and gbb, or GFP-tagged Dpp and Gbb, revealing specific effects of Dpp on growth. C. dppblk-GAL4 drives expression of a UAS-coupled nuclear GFP exclusively in SCs of the AG. D, E. Mosaic expression of dpp-RNAi or Dpp-GFP in a subset of SCs has a stronger effect on nuclear growth in expressing cells (on–green arrows) than in non-expressing (off–red arrows; white dashed circle) SCs, although dpp knockdown also reduces growth in the latter. F-K. Co-expression of dpp-RNAi with CD63-GFP using the dsx-GAL4 driver (G) reduces non-acidic SV number (eg., marked by arrowhead) and increases GFP fluorescence in largest MVBL (arrow; stained with Lysotracker Red) compared to controls (F); the statistical analysis of these changes for two independent RNAis is shown in H and I respectively. Knockdown of dpp either results in a small increase in the size of the largest MVBL or no significant size change (J), and reduces exosome secretion (K). Confocal images are from fixed glands (A, C, D) stained with DAPI (blue) and for Fas3 (yellow) or from living glands (F, G). Genotypes for images are: w; esg-GAL4 tub-GAL80ts UAS-FLP; UAS-GFPnls actin>FRT>CD2>FRT>GAL4/P[TRiP.HMS00011]attP2 (A and mosaic in D; the esgF/Ots driver was also used to generate data in E); w; P[w+ UAS-GFPnls]; P[w+ dppblk-GAL4] (C); w; UAS-CD63-GFP tub-GAL80ts; dsx-GAL4 combined with no other transgene (F) or P[TRiP.HMS00011]attP2 (III) (G). ***P<0.001, Kruskal-Wallis test, n = 10. Scale bar for A, D is 20 μm, F, G, 10 μm, and for C, 50 μm.</p

Cancer cells require survival strategies to respond to microenviromental changes and out-compete ... more Cancer cells require survival strategies to respond to microenviromental changes and out-compete their neighbours. They activate stress response mechanisms under extreme microenvironmental conditions, some of which are controlled by the amino acid-sensitive kinase complex, mechanistic Target of Rapamycin Complex 1 (mTORC1). Exosomes are secreted nanovesicles made inside intracellular endosomal compartments that mediate a specialised and complex form of intercellular signalling that can reprogramme target cells via the action of multiple active cargos. I investigated whether mTORC1 activity might modulate the type of exosome secreted in response to microenvironmental changes. Here I identify a new form of mTORC1-regulated exosome biogenesis and signalling involving recycling multivesicular endosomes (rMVEs), a previously unrecognised site for exosome biogenesis. Reduced activity of a specific form of glutamine-sensitive mTORC1 in HCT116 colorectal cancer cells results in an ‘exosome switch’ in which exosomes are preferentially released from these compartments instead of late endosomes. Importantly, RAB11a is found in association with at least a proportion of rMVEs that generate these alternative exosomes and is loaded on to some of their ILVs, providing a RAB signature of compartmental origin. I provide evidence that this exosome switch is conserved in other cancer cell types. My study also presents a proteomics analysis of extracellular vesicle (EV) preparations from normal and mTORC1-inhibited cells. I demonstrate that EV preparations isolated following exosome switching have enhanced pro-angiogenic properties and novel tumour growth-promoting activities. Activation of the receptor tyrosine kinase c-MET and its downstream mitogen-activated protein kinase (MAPK) ERK via phosphorylation is stimulated by these EVs, providing a potential explanation for their growth-promoting effects. Subsequent studies in the lab have demonstrated that several of these pro-tumorigenic activities are mediated by exosomes. I conclude that stress-induced mTORC1 inhibition allows tumour cells to initiate a novel exosome secretion pathway that potentially mediates a cancer cell survival plan that reverses microenvironmental change and supports tumour adaptation. In the future, blocking this response could improve patient outcome following treatment with mTORC1-inhibitory or anti-angiogenic drugs that have currently met with limited success in the clinic.</p

Molecular Biology of the Cell, 2016

The American Journal of Human Genetics, 2021

Genome-wide association studies (GWASs) have discovered 20 risk loci in the human genome where ge... more Genome-wide association studies (GWASs) have discovered 20 risk loci in the human genome where germline variants associate with risk of pancreatic ductal adenocarcinoma (PDAC) in populations of European ancestry. Here, we fine-mapped one such locus on chr16q23.1 (rs72802365, p = 2.51 × 10-17, OR = 1.36, 95% CI = 1.31-1.40) and identified colocalization (PP = 0.87) with aberrant exon 5-7 CTRB2 splicing in pancreatic tissues (pGTEx = 1.40 × 10-69, βGTEx = 1.99; pLTG = 1.02 × 10-30, βLTG = 1.99). Imputation of a 584 bp structural variant overlapping exon 6 of CTRB2 into the GWAS datasets resulted in a highly significant association with pancreatic cancer risk (p = 2.83 × 10-16, OR = 1.36, 95% CI = 1.31-1.42), indicating that it may underlie this signal. Exon skipping attributable to the deletion (risk) allele introduces a premature stop codon in exon 7 of CTRB2, yielding a truncated chymotrypsinogen B2 protein that lacks chymotrypsin activity, is poorly secreted, and accumulates intracellularly in the endoplasmic reticulum (ER). We propose that intracellular accumulation of a nonfunctional chymotrypsinogen B2 protein leads to ER stress and pancreatic inflammation, which may explain the increased pancreatic cancer risk in carriers of CTRB2 exon 6 deletion alleles.

Developmental Cell, 2019

Highlights d Serum LPA signaling through PI3K/Akt regulates the Rab11-Rab8 ciliogenesis cascade d... more Highlights d Serum LPA signaling through PI3K/Akt regulates the Rab11-Rab8 ciliogenesis cascade d Akt phosphorylates Rabin8 near its GEF domain, affecting Rab8-dependent cilia assembly d Akt stabilizes Rab11-effector binding to WDR44 by phosphorylating WDR44 at Ser342/344 d Effector switch from Rab11-WDR44 to Rab11-FIP3 initiates Rabin8 preciliary trafficking

PLOS Genetics, 2016

Oncogene, 2015