Direct RNA sequencing mediated identification of mRNA localized in protrusions of human MDA-MB-231 metastatic breast cancer cells

Jakobsen, Kristine Raaby; Sørensen, Emilie; Brøndum, Karin Kathrine; Daugaard, Tina Fuglsang; Thomsen, Rie; Nielsen, Anders Lade

doi:10.1186/1750-2187-8-9

Cited by 23 publications

(31 citation statements)

References 57 publications

(98 reference statements)

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“…The calculated RPKM values thereby represent the relative abundance of a given mRNA in the total number of mapped sequences. We selected mRNAs with RPKM values larger than 5 in both Ps and CB for downstream analysis, to eliminate insignificant results in the calculation of ratios [22] . The total number of mRNAs with RPKM values larger than 5 in both Ps and CB was 7591 (Supplementary Table S1).…”

Section: Resultsmentioning

confidence: 99%

Protrusion-localized STAT3 mRNA promotes metastasis of highly metastatic hepatocellular carcinoma cells in vitro

et al. 2016

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Aim: Recent evidence shows that localization of mRNAs and their protein products at cellular protrusions plays a decisive function in the metastasis of cancer cells. The aim of this study was to identify the variety of proteins encoded by protrusion-localized mRNAs and their roles in the metastasis and invasion of liver cancer cells. Methods: Highly metastatic hepatocellular carcinoma cell line HCCLM3 and non-metastatic hepatocellular carcinoma cell line SMMC-7721 were examined. Cell protrusions (Ps) were separated from cell bodies (CB) using a Boyden chamber assay; total mRNA population in CB and Ps fractions was analyzed using high-throughput direct RNA sequencing. The localization of STAT3 mRNA and protein at Ps was confirmed using RT-qPCR, RNA FISH, and immunofluorescence assays. Cell migration capacity and invasiveness of HCCLM3 cells were evaluated using MTT, wound healing migration and in vitro invasion assays. The interaction between Stat3 and growth factor receptors was explored with co-immunoprecipitation assays. Results: In HCCLM3 cells, 793 mRNAs were identified as being localized in the Ps fraction according to a cut-off value (Ps/CB ratio) >1.6. The Ps-localized mRNAs could be divided into 4 functional groups, and were all closely related to the invasive and metastatic properties. STAT3 mRNA accumulated in the Ps of HCCLM3 cells compared with non-metastatic SMMC-7721 cells. Treatment of HCCLM3 cells with siRNAs against STAT3 mRNA drastically decreased the cell migration and invasion. Moreover, Ps-localized Stat3 was found to interact with pseudopod-enriched platelet-derived growth factor receptor tyrosine kinase (PDGFRTK) in a growth factordependent manner. Conclusion: This study reveals STAT3 mRNA localization at the Ps of metastatic hepatocellular carcinoma HCCLM3 cells by combining application of genome-wide and gene specific description and functional analysis.

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Section: Resultsmentioning

confidence: 99%

Protrusion-localized STAT3 mRNA promotes metastasis of highly metastatic hepatocellular carcinoma cells in vitro

et al. 2016

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“…This not only suggests a transcript-specific role for the asymmetric RAB13 mRNA distribution but also offers an excellent model transcript to study mRNA localisation in angiogenesis. Similarly to endothelial cells, the presence of RAB13 mRNA at the leading edge has been previously reported in diverse cell types (Jakobsen et al, 2013;Mili et al, 2008;Zappulo et al, 2017), suggesting that the localisation of these transcripts may be part of the conserved machinery regulating cell migration. Nevertheless, a detailed understanding of the mechanisms of mRNA localisation and their implications in complex morphogenetic processes had yet to be addressed.…”

Section: Discussionmentioning

confidence: 99%

“…To start investigating the implications of this biological mechanism in endothelial cell migration and angiogenesis, we carried out a series of assays culturing Human Umbilical Vein Endothelial Cells (HUVECs) in modified Boyden chambers (Boyden, 1962) (hereafter referred as Transwells). This experimental setup allows the leading edge of migrating cells to extend protrusions through porous membranes separating two media-containing chambers and has been widely used to isolate compartmentalised mRNAs from diverse cell types (Jakobsen et al, 2013;Mili et al, 2008;Stuart et al, 2008;Zappulo et al, 2017). HUVECs were cultured for 2 hours in the upper chambers of Transwells containing 3µm pore membranes, in low serum conditions.…”

Section: Resultsmentioning

confidence: 99%

“…In this context, RAB13-mediated trafficking delivers the GEF Syx to the cell front, where together with its substrate RhoA modulates endothelial cell migration (Wu et al, 2011). Although RAB13 mRNA has been previously identified in protrusions formed at the front of migrating fibroblasts and tumour cells (Jakobsen et al, 2013;Mardakheh et al, 2015;Mili et al, 2008), the in-vivo morphogenetic function of the intracellular targeting of RAB13 mRNA (or indeed any other mRNA) is currently unknown.…”

Section: Introductionmentioning

confidence: 99%

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mRNA compartmentalisation spatially orients tissue morphogenesis

Costa

Bradbury

Tarannum

et al. 2018

Preprint

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SUMMARYThe subcellular distribution of mRNAs is a fundamental biological mechanism implicated in the spatial regulation of gene expression in many cellular contexts. However, whether mRNA localisation contributes to complex morphogenetic processes underpinning tissue development remains unknown.We focused on a model transcript, RAB13, and demonstrated how it is preferentially localised to and locally translated at the leading edge of migrating endothelial cells. We generated a novel MS2-GFPnls zebrafish reporter strain and use the 3' UTR of RAB13 to show, for the first time, tissuespecific visualisation of a localised RNA in migratory cell processes in a living vertebrate organism.Moreover, after mutating the endogenous 3' UTR of RAB13 to disrupt transcript localisation in-vivo, we report altered formation of endothelial cell protrusions within sprouting vessels. Hence, we demonstrate the involvement of mRNA localisation in endothelial cell migration during angiogenesis and are the first to reveal that subcellular transcript distribution modulates tissue morphogenesis.

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“…Rab13 mRNA is enriched in pseudopodial protrusions in MCF7 and MDA-MB-231 cancer cell lines and migratory fibroblasts (39,40). The 3Ј-untranslated region of Rab13 is necessary and sufficient to direct Rab13 mRNA to cellular protrusions in response to migratory cues (40).…”

mentioning

confidence: 99%

Regulation of Cancer Cell Behavior by the Small GTPase Rab13

Ioannou

McPherson

2016

Journal of Biological Chemistry

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The members of the Rab family of GTPases are master regulators of cellular membrane trafficking. With ϳ70 members in humans, Rabs have been implicated in all steps of membrane trafficking ranging from vesicle formation and transport to vesicle docking/tethering and fusion. Vesicle trafficking controls the localization and levels of a myriad of proteins, thus regulating cellular functions including proliferation, metabolism, cellcell adhesion, and cell migration. It is therefore not surprising that impairment of Rab pathways is associated with diseases including cancer. In this review, we highlight evidence supporting the role of Rab13 as a potent driver of cancer progression.The members of the Rab (Ras-related in brain) family of proteins are master regulators of vesicle trafficking. They are evolutionarily conserved with ϳ70 members in humans, and each Rab is localized predominantly to a specific intracellular organelle or vesicle (1). Rabs have been implicated in every step of vesicle trafficking including the formation of cargo-laden membrane vesicles and tubules, transport of these carriers on the cytoskeleton, tethering and docking of the carriers at the target membrane, and finally membrane fusion and delivery of cargo. Not surprisingly, functional impairment of Rab pathways is associated with diseases including immunodeficiencies, neurological disorders, and cancer (2). One such Rab, Rab13, controls cellular functions that are often altered in cancer, and Rab13 is now recognized as an important driver of cancer progression. Here, we discuss the importance of Rab13 in the physiology of cancer with a focus on Rab13 trafficking pathways contributing to tumorigenicity. The Biology of Rab13As for all small GTPases, Rab13 cycles between an active, GTP-bound state and an inactive, GDP-bound state. Activation of Rab13 is mediated by DENND2B and DENND1C/connecdenn 3, guanine nucleotide exchange factors (GEFs) 2 that interact with GDP-bound Rab13 and facilitate the exchange of GDP for GTP (3-5). These proteins each bear a differentially expressed in normal and neoplastic cells (DENN) domain, an evolutionarily ancient protein module that encodes GEF activity for a large number of Rab GTPases (6 -8). Rab13 is inactivated by the GTPase-activating protein (GAP) Akt substrate 160 (AS160)/TBC1D4, which enhances the ability of Rab13 to hydrolyze GTP (3-5). In its active form, Rab13 binds to several effectors including MICAL-L1, MICAL-L2, and PKA (9, 10). Rab13, which is ubiquitously expressed, is thought to have diverged from its closest homologue Rab8, early in the vertebrate lineage (11). Similar to Rab8, Rab13 has been implicated in both biosynthetic and endosomal recycling pathways. Rab13 is found at the trans-Golgi network, on recycling endosomes, on late endosomes, and at the plasma membrane (12-14). Disruption of Rab13 function inhibits the delivery of cargo that reaches the plasma membrane via endosomes from either the biosynthetic or the endocytic recycling pathways. Thus, Rab13 is thought to control membrane t...

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Direct RNA sequencing mediated identification of mRNA localized in protrusions of human MDA-MB-231 metastatic breast cancer cells

Cited by 23 publications

References 57 publications

Protrusion-localized STAT3 mRNA promotes metastasis of highly metastatic hepatocellular carcinoma cells in vitro

Protrusion-localized STAT3 mRNA promotes metastasis of highly metastatic hepatocellular carcinoma cells in vitro

mRNA compartmentalisation spatially orients tissue morphogenesis

Regulation of Cancer Cell Behavior by the Small GTPase Rab13

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