Direct RNA sequencing mediated identification of mRNA localized in protrusions of human MDA-MB-231 metastatic breast cancer cells
© Jakobsen et al.; licensee BioMed Central Ltd. 2013
Received: 22 March 2013
Accepted: 20 August 2013
Published: 1 September 2013
Protrusions of cancer cells conferrers a vital function for cell migration and metastasis. Protein and RNA localization mechanisms have been extensively examined and shown to play pivotal roles for the functional presence of specific protein components in cancer cell protrusions.
To describe genome wide RNA localized in protrusions of the metastatic human breast cancer cell line MDA-MB-231 we used Boyden chamber based methodology followed by direct mRNA sequencing.
In the hereby identified group of protrusion localized mRNA some previously were described to be localized exemplified by mRNA for Ras-Related protein 13 (RAB13) and p0071 (Plakophilin-4/PKP4). For other transcripts, exemplified by mRNA for SH3PXD2A/TKS5 and PPFIA1/Liprin-α1, only the corresponding proteins previously were described to have protrusion localization. Finally, a cohort of MDA-MB-231 protrusion localized transcripts represents novel candidates to mediate cancer cell subcellular region specific functions through mRNA direction to protrusions. We included a further characterization of p0071, an armadillo repeat protein of adherence junctions and desmosomes, in MDA-MB-231 and non-metastatic MCF7 cells including analysis of novel identified alternative spliced p0071 mRNA isoforms. The results showed isoform and cell type specific p0071 mRNA localization.
Altogether, the presented data represents a genome wide and gene specific descriptive and functional analyses of RNA localization in protrusions of MDA-MB-231 metastatic cancer cells.
A metastasizing cancer cell is abridged to essential challenges: the ability to escape from the tumour and migrate to the blood vessels; survive in the circulation; make an entrance in another tissue; and the capability of surviving and growing in the new environment . A metastasising cancer cell arising from epithelial tissue is characterized by the gain of mesenchymal properties involving increased motility in a transition that equals the epithelial-mesenchymal transition (EMT) seen during development . Epithelial cells function as a barrier to establish and maintain an internal and an external environment and are immobile with a clear apico-basal polarity. Mesenchymal cells on the other hand are mobile, have more transient adhesive properties, are more responsive to extracellular attractants directing the movements, are capable of invading the basement membrane, and without apico-basal polarity . Migration can occur as a response to variation of the stimulation from the extracellular environment, such as gradients of chemokines and extracellular matrix proteins. The cancer cell migration process is dynamic with attachments and detachments to the extracellular matrix resulting in movement . This is mediated by cellular adhesion molecules such as integrins and cadherins and conformational cytoskeletal changes with resulting pseudopodial dynamics . Integrins have different affinities for the extracellular matrix proteins and may activate and respond to different signalling pathways. In general, integrins have a high expression at the cellular leading edge and serve to stabilize pseudopodia through mediating contacts between the cytoskeleton and extracellular matrix (ECM) proteins [4, 5]. The ability to form pseudopodial protrusions is closely related to cancer cell invasive and metastatic properties [6, 7]. A core for pseudopodia formation is generated at the ventral surface of the cell by dendritic and bundled actin networks . Actin polymerization and contractility are mediated by members of the RHO family of small GTP-binding proteins, including RAC, RHO and CDC42, activated by integrin interactions [9, 10]. The actin core grows by elongation of actin bundles denoted filopodia . If the actin dendritic network (lamellipodia) advances as fast as the actin bundles elongate, the structure of the elongated invadopodium appears as a microspike or roots of filopodia. Elongation of pseudopodia beyond 5 μm is permitted by the infiltration of microtubules. Intermediate filaments are entering in mature pseudopodia and have an impact only in later stages of invasion .
Asymmetric localization of RNA is involved in cell polarisation and pseudopodial formation [11, 12]. RNA localization depends on motor protein movements on the microtubules and the actin filaments of the cytoskeleton [11, 13]. Moreover, RNA localization depends on a dynamic interaction between cis-elements within the RNA and associated trans-factors forming localizing RNA protein complexes. A well-studied example is ZBP1 which is a RNA-binding trans-factor for RNA localization involved in generating apico-basal polarity and repressed in metastasizing breast cancer cells . ZBP1 associates with the zip-code cis-element present in the 3′-UTR of the β-actin mRNA and mediates transport of the complex to the leading edge dependent on an intact actin cytoskeleton [14, 15]. The RNA cis-elements for localization can either be directly sequence dependent as the zip-code or structural dependent. Several studies have addressed the identity of mRNA and protein with localization to pseudopodia in neurons, astrocytes, fibroblasts, and cancer cells [16–21]. Proteome and array based transcriptome analyses of metastatic tumour cell lines have resulted in the identification of pseudopodia localized proteins involved in pseudopodia formation, actin cytoskeleton dynamics, and cell migration and invasion .
The current knowledge concerning mRNA localization to metastatic cell pseudopodial protrusions is not yet fully comprehensive. We took advantage of a recently presented high throughput next generation sequencing method, direct RNA sequencing (DRS), which is a highly sensitive and low bias approach to identify and quantify total mRNA [23–25]. RNA localized in metastatic breast cancer cell pseudopodial protrusions was purified from the metastatic human breast cancer cell line MDA-MB-231 using a refined Boyden chamber assay [18, 26–28]. Poly-adenylated mRNA molecules isolated from MDA-MB-231 cell protrusions and cell bodies were analyzed by DRS and protrusion localized mRNA determined. The presented procedure is an alternative method for identification of pseudopodial protrusion localized RNA and can contribute to broaden the overall understanding of the involvement of sub-cellular mRNA localization in cancer cell metastasis.
Materials and methods
Boyden chamber assay for cell protrusion isolation and DRS
Human breast cancer MDA-MB-231 and MCF7 cells were purchased from the American Tissue Culture Collection (ATCC). Before experimental procedures, cells were passaged once using 0.5% trypsin-EDTA (GIBCO). Cells were grown in DMEM (GIBCO) with 10% fetal calf serum (FCS), glutamine, penicillin, and streptomycin at 37°C in 5% CO2 humidified atmosphere .
A modified cell protrusion isolation method was used [26, 28]. For the Boyden chamber assay we used 9.6 cm2 (6-well format) cell culture inserts (BD Falcon) with a 1 μm pore size uncoated polystyrene membrane. Membranes were coated with the ECM proteins collagen type-I (Sigma C7661) in a 10 μg/mL final concentration incubating at 37°C. for 2 h. Afterwards the membranes were directly transferred to a 6-well tissue culture dish containing serum free DMEM. Cells were serum starved overnight and detached by 0.5% Trypsin-EDTA, and the trypsin was inactivated by suspending cells in DMEM containing 10% FCS. Cells were pelleted and re-suspended in serum free medium, and 2 × 106 cells were transferred to one membrane insert. Cell protrusions were allowed to grow through the membrane for 24 h before protein and RNA purification.
For RNA and protein purification inserts were washed in PBS and cell protrusions were harvested using a sharp cell scraper (Costar). The lower side of the membrane was scraped first and the cell scraper thoroughly washed in 1 ml TRI-Reagent (Sigma) for RNA or in 1 ml 1x loading buffer (Fermentas) for protein. It was carefully checked that the cell scraper was only used once on the membrane in order to avoid cell lysis by the TRI-Reagent or loading buffer, which could have resulted in contamination of the protrusion fraction of lysed cells from the upper-side. Cells on the upper side was subsequently scraped and transferred to TRI-Reagent or loading buffer. RNA was precipitated using 1 μg glycogen. Single RNA molecules were sequenced by DRS  using the Helicos Biosciences platform (Helicos Biosciences, Boston, MA). DRS was performed once with pools of RNA representing CF or PF from six independent Boyden chamber inserts. Equal amounts of RNA isolated from CF or PF were used for input in DRS. Bioinformatics raw data analysis and sequence alignment was made by Helicos Biosciences.
RNA visualization and immunofluorescence
Total cytosolic RNA was visualized by ethidium bromide (EtBr) essential as previously described . Briefly, MDA-MB-231 cells were prepared as described under Boyden Chamber assay. RNase control cells were made by treating membranes with 125 ng/μL RNase for 30 minutes. RNase was inactivated by vanadyl-ribonucleoside complex for 10 minutes. Membranes were washed twice with PBS, cells permeabilized in Triton X-100 0.5% for 10 min and subsequently washed with PBS. 1 μM EtBr in PBS was added for 15 min, cells washed twice with PBS and DAPI stained for 2 min. After washing in PBS, half of the cell material on the upper side of the membrane was wiped away. Membranes were sliced and in both orientations fixated in a drop of Prolong GOLD mounting media (Invitrogen) in a petri dish and analysed by microscopy.
For immunofluorescence analysis MDA-MB-231 or MCF7 cells were grown to ~70% confluence on 0.17 mm thick cover slips (Marienfeld), fixed in 4% paraformaldehyde in PBS (Electron Microscopy Sciences) for 20 min at room temperature, and permeabilized in 0.5% tritonX-100 (Sigma). Cells were incubated with primary antibody dissolved in blocking buffer for 1 h at room temperature, and were then treated with secondary antibody in blocking buffer for 45 min at room temperature. Cell nuclei were stained with DAPI and the coverslips were mounted. In immunofluorescence analysis of Boyden chamber membranes the cell bodies were removed from one half of the upper-side of the membrane. Membranes were incubated with antibodies as described above. Instead of cutting out the membrane and mounting, the Boyden chamber inserts were kept in PBS for visualization. For immunofluorescence rabbit anti-α-tubulin 1:500 (Rockland), anti-p0071 1:10 (Progen) and secondary antibody Alexa 488 conjugated goat anti-rabbit IgG 1:2000 (Invitrogen), Alexa 555 conjugated donkey anti-rabbit IgG 1:2000 (Invitrogen) or Alexa 555 conjugated goat anti-mouse IgG 1:2000 (Invitrogen) was used. All images for IF were made on a Zeiss axiovert 200 m microscope, with a plan apochromatic 63x 1.4 NA objective, a HBO 100 W mercury light source, and a CoolSNAP-HQ cooled CCD camera (Photometrics) operated by MetaMorph®. We took z-stacks with 20 sections 0.2 μm step size and 500 ms exposure. For the shown immunofluorescence images a single, best in focus, 2D image was selected from a 20 sections z-stack.
Reverse transcriptase, PCR and RT-qPCR
cDNA was made from total RNA by iScript cDNA synthesis kit (Bio-Rad) containing both random hexamer and oligo(dT) primers using 1 μL purified total RNA solution per reaction. PCR was performed under standard conditions . Quantitative real time PCR (RT-qPCR) was performed using SYBR Green 480 master mix (Roche), and reactions were run on a Roche Lightcycler™ 480, with a primer annealing temperature of 58°C. PCR products were verified by gel electrophoresis and melting curve peaks. Primer sequences are shown in Additional file 1: Table S1. RT-qPCR amplifications were made in triplicates for each gene and the Ct values were converted into linear values using the Xo method . Student’s 2-Tailed t-test was performed accordingly to (http://studentsttest.com). The localization ratio was determined as the ratio between the mean expression in the cell body fraction and the protrusion fraction. RT-qPCR experiments to verify DRS results were performed on Boyden chamber purified RNA samples purified and processed independently of the RNA samples used for DRS. For p0071 mRNA isoform detection the primer sequences are shown in Additional file 1: Table S1.
Proteins were separated by SDS-PAGE in a 4-15% gradient polyacryl amide gel (Bio-Rad). Proteins were transferred to a nitrocellulose membrane which was cut in two parts. The following primary antibodies were used for protein detection: Anti Histone H3 (1:10000, Rabbit, Abcam ab1791, 19 kDa); Anti α-Tubulin (1:5000, Rabbit, Rockland 600-401-880, 51 kDa), Anti β-Actin (1:2500, Rabbit, Sigma A2103, 42 kDa); Anti Zeb1 (1:500, Rabbit, Sigma HPA027524, 200 kD); Anti ANP32B (1:100, Rabbit, Sigma SAB4500125, 30 kDa). Antibody procedures and detection by horse radish peroxidase (HRP) conjugated antibody (Dako, 1:10000) were performed as previously described [28, 29].
Results and discussion
RNA isolation from MDA-MB-231 cell protrusions
For RNA isolation MDA-MB-231 cell protrusions were separated from cell bodies by the Boyden chamber assay and total RNA from PF and CF was isolated for downstream analysis. Material was sampled from 6 independent Boyden chambers each containing 2 × 106 cells. By quantitative real-time PCR (RT-qPCR) we monitored RNA quantity and quality by examining RNA previously determined to localize in MDA-MB-231 cell protrusions. RT-qPCR was performed on cDNA made from equal RNA concentrations from CF and PF. We included primers against β-Actin (ACTB), ARPC3, RAB13, KANK2/ANKRD25, RHOA, Vimentin (VIM), GAPDH and Cortactin (COR). RAB13 and KANK2 mRNA were in a previous study shown to be localized in protrusions of NIH3T3 cells . RHOA, COR and ACTB are all involved in the dynamics of the actin skeleton, VIM is an intermediary filament protein with an expression correlating with the mesenchymal phenotype and is up-regulated during EMT, and ARPC3 and GAPDH represents housekeeping genes. ARPC3 mRNA was previously determined to have uniform cytoplasmic localization in mouse astrocytes and NIH3T3 cells and chosen as reference gene [16, 28]. We observed localization for RAB13 (14-fold) and KANK2 (2-fold) mRNA compared to the used normalization control ARPC3 mRNA whereas no localization was observed for VIM, COR, RHOA and ACTB (Figure 2C). Altogether the RT-qPCR analyses pointed towards that RNA isolated from MDA-MB-231 protrusions is in a quality and quantity which allows for further biochemical characterization.
Direct RNA sequencing (DRS) of MDA-MB-231 protrusion and cell body mRNA
MDA-MB-231 protrusion localized mRNA
Annotation analysis of MDA-MB-231 protrusion localized mRNA
We selected the top 100 most protrusion localized mRNA for annotation analysis using the IPA Ingenuity online platform. For reference we also analyzed a list of all transcripts determined from DRS with ≥ 5 tpm. The most striking outcome was 4 functional pathways overrepresented in the group of protrusion localized mRNA: Cellular assembly and organization; Protein synthesis and modification; Cell movement; and Cancer (Figure 3B). More surprisingly, functional pathway analyses and manual inspection pointed that the localized mRNA included several transcripts encoding nuclear proteins exemplified by NET1 (ratio 4.6, NM_005863), ANP32B (ratio 5.6, NM_ 006401), ZEB1 (ratio 8.4, NM_001128128) and CENPB (ratio 6.5, NM_001810) (Table 1 and Figure 3C). Transcripts for nuclear proteins were also identified in other studies describing protrusion localized mRNA and for example NET1 mRNA was previously described to localize to the basement membrane in EMT during gastrulation [16, 22, 32]. In MDA-MB-231 cells NET1 controls focal adhesion kinase activation during cell movement and motility during ECM invasion in a RAC1 dependent extranuclear localization dependent manner . ZEB1 protein is highly expressed in MDA-MB-231 cells and is a transcription factor involved in the regulation of breast cancer EMT, cell motility and metastasis . An involvement of ANP32B and CENPB proteins in breast cancer cell migration and metastasis is to our knowledge not yet described but we note that elevated ANP32B expression is associated with poor prognosis in breast cancer . RT-qPCR verified the mRNA localization in protrusions for NET1, ANP32B, ZEB1 and CENPB (Figure 3C). Western blot analysis using CF and PF protein extracts normalized for α–Tubulin showed that in the PF ZEB1 and ANP32B were not detectable in agreement with in preference nuclear localization of the proteins (Figure 3D). We note that the minute amounts of protein material isolable from PF and the according high dilution of the CF sample to equalize for α–Tubulin expression level in the two samples hindered conclusive Western blot analysis of a large set of proteins assayed including for example NET1 and p0071 due to either an insufficient expression level or antibody sensitivity.
Shankar et al. previously presented the identification of protein and mRNA localizing to protrusions of different metastatic cancer cell lines including MDA-MB-231 cells . mRNA localization was addressed by microarray studies of RNA purified from protrusions resulting in identification of 384 transcripts determined to have a protrusion localization ratio above the threshold value 1.6 . Of these transcripts Desmoyokin (AHNAK nucleoprotein), Septin-9 and Calgizzarin (S100A11) were shown to encode proteins involved in cell migration and invasion . We note that by DRS we find mRNA localization in protrusions of Desmoyokin (mRNA localization ratio 1.62, NM_024060) and Calgizzarin (mRNA localization ratio 2.1, NM_005620) whereas Septin-9 (ratio 1.1, NM_0011134914) mRNA localization was absent. The approximately 2-fold increase in the total number of identified protrusion localized mRNA by the hereby presented DRS approach could be a consequence of higher sensitivity and shows the importance of using different experimental approaches. Several of the mRNA species within the DRS mRNA list encode proteins with a described function in protrusions and cell migration processes. However, the participation of mRNA localization to confer the final protein localization has not been described for the majority of these transcripts. In the following we will give few examples of metastatic breast cancer relevant transcripts, which to our knowledge has not been attributed before with mRNA localization. The mRNA localization of this subgroup in cell protrusions was confirmed by RT-qPCR (Figure 3C). SH3PXD2A/TKS5/FISH (mRNA localization ratio 8.1, NM_014631) is a SH3 domain-rich protein which is an adaptor protein in clustering structural and enzymatic components of invadopodia [36, 37]. SH3PXD2A function is required for invadopodia formation, for degradation of the extracellular matrix, and for cancer cell invasion [36, 37]. The trafficking kinesin protein TRAK2 (mRNA localization ratio 9.2, NM_015049) is a mediator of axonal mitochondrial transport and is a kinesin adaptor for mitochondria at the tips of cellular processes . PPFIA1/Liprin-α1 (mRNA localization ratio 4.1, NM_003626) adaptor protein is a regulator of cell edge dynamics during motility . In MDA-MB-231 cells PPFIA1 is required for migration and invasion by mediating ECM degradation and the formation of lamellipodia and invadopodia .
Characterization of p0071 mRNA in MDA-MB-231 and MCF7 cells
p0071 is associated with different steps in carcinogenesis [40, 43, 53, 54]. However, p0071 mRNA localization has not been characterized in cancer cells and this was pursued in this study. Microtubule transport of p0071 and RAB13 mRNA to the cellular leading edge is dependent on Adenomatous polyposis coli (APC) protein which play a role in the anchoring of mRNA at the end of the microtubules . The loss of APC, which like Folliculin is a tumour-suppressor protein, is an initiating event in colorectal cancer . APC mRNA had a localization ratio of 1.4 in the DRS analyses which is similar to ARPC3 mRNA indicating lack of protrusion localization. We also note the lack of Delta-catenin and Folliculin mRNA localization in MDA-MB-231 cell protrusions examined by DRS (ratio 0.5 and 0.7, respectively).
Localization analysis by immunofluorescence showed the presence of p0071 protein throughout the cytoplasm of MDA-MB-231 cells with mostly perinuclear localization but also localization in some cell protrusions (Figure 4B). We note that in the immunofluorescence analysis approximately 50% of the MDA-MB-231 cells displayed distinct protrusions and in approximately 50% of these p0071 protein was detectable (Figure 4B). α-Tubulin showed a uniform localization in MDA-MB-231 cells (Figure 4B). To determine the localization of p0071 protein in non-metastatic breast cancer cells we examined the MCF7 cell line with epithelial-like cell morphology (Figure 4B). In MCF7 cells p0071 protein localization was most prominent to cell-cell contact surfaces in agreement with previous descriptions of p0071 protein localization to desmosomes and adherence junctions of contacting epithelial cells (Figure 4B) . The specificity of the p0071 antibody was verified by transfecting MDA-MB-231 cells with siRNA which resulted in p0071 depletion but no observable differences in protrusion forming capability and cell migration (data not shown).
Subcellular mRNA localization is mediated by a combination of mRNA cis-elements, as for example the Zip-code and G-rich sequences often residing in the 3′-UTR, which associates with trans acting localization factors [11, 13]. We note that the mRNA/mRNP transport protein FMRP previously was described to target p0071 mRNA [16, 55, 56]. Inspection of the p0071 gene at UCSC Genome Browser (NCBI36/hg18 assembly) showed the presence of putative alternative splicing and poly-adenylation events consequently generating p0071 mRNA isoforms with different coding regions and 3′-UTR sequences. Schematic description of the hereby examined four putative p0071 mRNA isoforms is shown in Figure 5C. p0071-1 (transcript variant 1) includes exon 19 and encodes a protein of 1192 amino acids. p0071-2 (transcript variant 2) lacks exon 19 (length 129 base pairs) and as consequence results in an internal p0071 protein deletion of 43 amino acids with a resulting p0071 protein isoform of 1149 amino acids. The alternative poly-adenylated p0071-3 and p0071-4 mRNA isoforms include an extended exon 19 sequence, 19a, and include or exclude an alternative spliced exon, 20a, respectively (Figure 5C). Both events result in p0071 protein isoforms with distinct C-terminal sequences including 1112 amino acids for p0071-3 and 1128 amino acids for p0071-4. The four p0071 protein isoforms include all ten armadillo ARM repeats. We also note the existence of complex alternative splicing and poly-adenylation of delta-catenin transcripts . p0071 mRNA expression analysis using primer combinations detecting the four putative p0071 mRNA isoforms in MCF7 and MDA-MB-231 cells, together with a primer set spanning from exon 6 to exon 7 and accordingly envisaged to detect all the 3′-region variants of p0071 (p0071-pan) mRNA, showed expression of all four isoforms and cell type dependent p0071 alternative splicing and poly-adenylation (Figure 5D and data not shown). Exon 18 splicing to exon 19 compared with exon 18 splicing to exon 20 was relatively increased in MCF7 cells resulting in increased expression levels of the exon 19 including p0071-1, p0071-3 and p0071-4 mRNA isoforms (Figure 5D). We note that albeit our RT-qPCR analyses are not absolutely quantitative the analyses indicate that p0071-1 and p0071-2 mRNA are expressed in similar order of magnitudes. p0071-3 mRNA is expressed to an approximately ten-fold lower level than p0071-1 and p0071-2 mRNA, and p0071-4 mRNA is expressed approximately 40-fold lower, which is close to the limit of detection particular in MDA-MB-231 cells. Boyden chamber analysis showed that all p0071 mRNA isoforms lacked significant protrusion localization in MCF7 cells (Figure 5B). In MDA-MB-231 cells p0071-1 and p0071-2 mRNA have protrusion localization ratios comparable with p0071-pan (Figure 5B). The p0071-3 mRNA protrusion localization ratio was close to 1 indicating localization in neither MDA-MB-231 nor MCF7 cells (Figure 5B). p0071-4 mRNA levels in MDA-MB-231 protrusions was limited to a level making absolute protrusion localization ratio calculations insignificant but indicates lack of localization (data not shown). Altogether, the p0071 mRNA analyses indicate that protrusions localization requires 3′-UTR sequences present in exon 21 and that p0071 mRNA protrusion localization have isoform and cell type specificity. The presented data are indicative of a model where localization of specific p0071 protein isoforms in MDA-MB-231 cells can be regulated at least partially through sub-cellular mRNA localization. If the observed p0071 mRNA localization to protrusions in MDA-MB-231 cells is mechanistically linked to down-regulation of desmosomes and adherent junctions during EMT and thereby contributing to breast cancer invasion and metastasis requires further analysis .
A limited amount of studies have addressed global-wide mRNA localization patterns in cancer cell protrusions. Here we show that a small size Boyden chamber assay can generate amounts of RNA from protrusions applicable for DRS. By this approach we identified hundreds of mRNA transcripts with localization in protrusions of metastatic breast cancer MDA-MB-231 cells. The presented approach can constitute an important experimental basis for additional studies to determine RNA localization as response to ECM-proteins and chemo-attractants. Moreover, the small amount of cellular material required for the DRS analyses will make the presented methodology highly applicable for studying the functional pathways mediating RNA localization using siRNA mediated depletion of candidate trans-factors and generation of cell lines with mRNA cis-element mutations. A more profound knowledge of the RNA localization to protrusions and how this localization is mediated can contribute considerable to the basic and clinical understanding of cancer cell metastasis.
This project was supported by Lundbeck Fonden, Arvid Nilssons Fond, Familien Hede Nielsens Fond, Andersen-Isted Fonden, Direktør Jacob Madsen og Hustru Olga Madsens Fond, Fabrikant Einer Willumsens Mindelegat, Marie og Børge Kroghs Fond, and Fonden til lægevidenskabens fremme (The A.P. Møller Foundation for the Advancement of Medical Science). KRJ and RT are supported by PhD fellowships from The Faculty of Health, Aarhus University, Denmark.
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