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A novel L1CAM isoform with angiogenic activity generated by NOVA2-mediated alternative splicing

  1. Francesca Angiolini
  2. Elisa Belloni
  3. Marco Giordano
  4. Matteo Campioni
  5. Federico Forneris
  6. Maria Paola Paronetto
  7. Michela Lupia
  8. Chiara Brandas
  9. Davide Pradella
  10. Anna Di Matteo
  11. Costanza Giampietro
  12. Giovanna Jodice
  13. Chiara Luise
  14. Giovanni Bertalot
  15. Stefano Freddi
  16. Matteo Malinverno
  17. Manuel Irimia
  18. Jon D Moulton
  19. James Summerton
  20. Antonella Chiapparino
  21. Carmen Ghilardi
  22. Raffaella Giavazzi
  23. Daniel Nyqvist
  24. Davide Gabellini
  25. Elisabetta Dejana
  26. Ugo Cavallaro  Is a corresponding author
  27. Claudia Ghigna  Is a corresponding author
  1. IEO, European Institute of Oncology IRCCS, Italy
  2. Consiglio Nazionale delle Ricerche, Italy
  3. University of Pavia, Italy
  4. Università degli Studi di Roma "Foro Italico", Italy
  5. Università degli Studi di Pavia, Italy
  6. FIRC Institute of Molecular Oncology, Italy
  7. The Barcelona Institute of Science and Technology, Spain
  8. Universitat Pompeu Fabra, Spain
  9. Institució Catalana de Recerca i Estudis Avançats, Spain
  10. Gene Tools LLC, United States
  11. IRCCS-Istituto di Ricerche Farmacologiche Mario Negri, Italy
  12. Karolinska Institutet, Sweden
  13. IRCCS San Raffaele Scientific Institute, Italy
  14. Uppsala University, Sweden
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Cite this article as: eLife 2019;8:e44305 doi: 10.7554/eLife.44305

Abstract

The biological players involved in angiogenesis are only partially defined. Here, we report that endothelial cells (ECs) express a novel isoform of the cell-surface adhesion molecule L1CAM, termed L1-ΔTM. The splicing factor NOVA2, which binds directly to L1CAM pre-mRNA, is necessary and sufficient for the skipping of L1CAM transmembrane domain in ECs, leading to the release of soluble L1-ΔTM. The latter exerts high angiogenic function through both autocrine and paracrine activities. Mechanistically, L1-ΔTM-induced angiogenesis requires fibroblast growth factor receptor-1 signaling, implying a crosstalk between the two molecules. NOVA2 and L1-ΔTM are overexpressed in the vasculature of ovarian cancer, where L1-ΔTM levels correlate with tumor vascularization, supporting the involvement of NOVA2-mediated L1-ΔTM production in tumor angiogenesis. Finally, high NOVA2 expression is associated with poor outcome in ovarian cancer patients. Our results point to L1-ΔTM as a novel, EC-derived angiogenic factor which may represent a target for innovative antiangiogenic therapies.

https://doi.org/10.7554/eLife.44305.001

eLife digest

Growing tumors stimulate the formation of new blood vessels to supply the oxygen and nutrients the cancerous cells need to stay alive. Stopping tumors from forming the blood vessels could therefore help us to treat cancer. To do so, we need to understand how different proteins control when and how blood vessels develop.

Cells make proteins by first ‘transcribing’ genes to form RNA molecules. In many cases, the RNA then goes through a process called alternative splicing. Proteins known as splicing factors cut out different segments of the RNA molecule and stick together the remaining segments to form templates for protein production. This enables a single gene to produce many different variants of a protein.

Angiolini, Belloni, Giordano et al. have now studied mouse and human versions of the cells that line the blood vessels grown by tumors. This revealed that a splicing factor called NOVA2 targets a protein called L1CAM, which is normally responsible for gluing adjacent cells together. Angiolini et al. found that NOVA2 splices L1CAM into a form not seen before. Instead of remaining anchored to cell surfaces, the newly identified form of L1CAM is released into the blood circulation, where it stimulates new blood vessels to grow.

Samples taken from the blood vessels of human ovarian tumors showed high levels of both NOVA2 and the modified form of L1CAM, while blood vessels in healthy tissue contain no, or very low levels of both proteins. Therefore, if the new form of L1CAM can be detected in the blood, it could be used to help cancer diagnosis, and to indicate which patients would benefit from treatments that restrict the growth of blood vessels in tumors. Further work is now needed to explore these possibilities.

https://doi.org/10.7554/eLife.44305.002

Introduction

L1CAM, also known as CD171 or L1, is a cell adhesion molecule encoded by the gene L1CAM. It is a cell surface glycoprotein with an extracellular portion that comprises six Ig-like domains, five fibronectin type III repeats, a short transmembrane (TM) domain, and a conserved cytoplasmic tail. A disintegrin and metalloproteinase (ADAM)10-mediated cleavage at membrane proximal site induces shedding of the L1CAM ectodomain (Mechtersheimer et al., 2001), while intramembrane processing mediated by γ-secretase generates a cytosolic domain which can translocate to the nucleus and modulate gene expression (Maretzky et al., 2005). L1CAM was initially identified in the nervous system and characterized for its important function in neural development and plasticity (Maness and Schachner, 2007). Further studies then showed that L1CAM is not restricted to the nervous system and, in particular, we reported its expression in the tumor vasculature of several cancer types, while no or very low L1CAM expression is detectable in normal vessels (Maddaluno et al., 2009; Magrini et al., 2014). L1CAM orchestrates different endothelial cell (EC) functions within tumor-associated vessels, such as permeability, pericyte coverage and polarity. Since these cellular processes influence tumor angiogenesis, cancer growth and metastasis, L1CAM has emerged as a potential target for tumor vascular-specific therapies (Magrini et al., 2014).

L1CAM occurs mainly in two alternatively spliced isoforms: while neurons typically express the full-length variant of L1CAM, non-neural cell types produce a shorter isoform which lacks exon 2 and exon 27. Exon 2 is involved in the interaction with other neuronal proteins, and exon 27 facilitates endocytosis of L1CAM (Schäfer and Altevogt, 2010).

Alternative splicing (AS) produces different mature transcripts (mRNAs) from a single primary pre-mRNA. AS decisions are modulated by a number of cis-acting motifs and splicing regulatory factors (SRFs) that function in a coordinate manner to promote or inhibit the inclusion of specific exons into the mRNA (Fu and Ares, 2014; Nilsen and Graveley, 2010). More than 90% of human protein-coding genes undergo AS (Pan et al., 2008; Wang et al., 2008) giving rise to different protein isoforms with distinct structural and functional properties. Hence, AS represents an important mechanism to expand the coding potential of the human genome, thus contributing to generate the cellular complexity of different tissue types and to support key functional properties (Chen and Manley, 2009; Baralle and Giudice, 2017). Notably, several findings highlighted a direct role of AS in promoting cancer progression (Anczuków and Krainer, 2016; Biamonti et al., 2014; Pradella et al., 2017). In particular, it has been shown that mutations or altered expression of specific SRFs allow neoplastic cells to generate cancer-specific AS isoforms involved in tumor establishment, progression and resistance to therapeutic treatments (Bonomi et al., 2013a; Anczuków and Krainer, 2016; Biamonti et al., 2014; Oltean and Bates, 2014). These ‘oncogenic AS switches’ can be used to stratify patients according to tumor stage (Stricker et al., 2017; Inoue and Fry, 2015), while their targeting represents a promising approach to improve the efficacy of anti-cancer treatments (Bonomi et al., 2013a; Agrawal et al., 2018; Anczuków and Krainer, 2016). However, in contrast to the established role of AS in tumor cells, it remains unclear whether this process is also relevant in tumor microenvironment and, in particular, in cancer vasculature. In fact, AS events specifically occurring in tumor-associated ECs have been described (Neri and Bicknell, 2005) and proposed as potential targets for antiangiogenic therapies (Steiner and Neri, 2011). However, how such AS events impact on the pathophysiology of tumor vasculature remains elusive.

Recently, we described the SRF Neuro-Oncological Ventral Antigen 2 (NOVA2) as a prominent regulator of AS during vascular development (Giampietro et al., 2015). NOVA2 was initially identified in neural cells where it controls AS of several genes involved in various neural developmental processes by binding to clusters of YCAY (Y = C/U) repeats within its pre-mRNA targets (Licatalosi et al., 2008; Ule et al., 2003; Zhang et al., 2010; Leggere et al., 2016; Saito et al., 2016). Our study revealed that NOVA2 is also expressed in vascular endothelium and is regulated during angiogenesis (Giampietro et al., 2015). NOVA2 controls at the post-transcriptional level the establishment of EC polarity, a process that is essential for vascular lumen formation and, hence, for angiogenesis (Iruela-Arispe and Davis, 2009). Accordingly, NOVA2 ablation causes defects in vascular lumen formation in vivo (Giampietro et al., 2015).

Here, we report a novel isoform of L1CAM expressed in ECs as the result of a NOVA2-induced AS event that removes the exon encoding the transmembrane domain of the protein. This gives rise to a soluble L1CAM variant, referred to as L1-ΔTM, that is released by ECs and is able to stimulate angiogenesis via autocrine/paracrine mechanisms. NOVA2 and L1-ΔTM are overexpressed in the vasculature of ovarian cancer and correlate with poor outcome and tumor vascularization, respectively. Our findings, therefore, implicate the novel NOVA2/L1-ΔTM axis in EC pathophysiology and in ovarian cancer aggressiveness.

Results

Alternative splicing of L1CAM in endothelium

We have recently reported the novel function of L1CAM in vascular endothelium (Magrini et al., 2014). Since AS is known to influence the biological activities of cell-surface adhesion molecules (Wang et al., 2005), it is possible that AS of L1CAM accounts for, or at least contributes to, its peculiar role in ECs. A bioinformatics analysis with the ExonMine program (http://www.imm.fm.ul.pt/exonmine/) (Mollet et al., 2010) identified a human expressed sequence tag (EST) in which the L1CAM exon 25 (a 135-nucleotide cassette exon) is excluded from the mature mRNA (Figure 1A). We then analyzed several normal human tissues and human ECs for the AS of human L1CAM exon 25 by RT–PCR (Figure 1B). In addition, we also investigated the AS of this exon in the mouse. In the murine gene, this exon is annotated as exon 26 by UCSC and Ensembl, due to the presence of an additional non-coding exon upstream of exon 1 (i.e., the one containing the ATG codon). Nevertheless, based on its high homology to the human L1CAM exon 25 (89% identity), we refer to it as exon 25 also in mouse L1cam. The AS of this exon was examined in normal mouse tissues, mouse EC lines and freshly purified murine ECs. As shown in Figure 1B and C, in both human and mouse samples the skipping of exon 25 mainly occurred in ECs. Overall, these data suggest that ECs express a novel alternatively spliced isoform of L1CAM devoid of exon 25.

Figure 1 with 1 supplement see all
Alternative splicing of L1CAM exon 25.

(A) 3’ region of human L1CAM gene and AS variants that are present as ESTs (data from UCSC Genome Browser). The green box indicates exon 25. The green arrow shows an EST with the skipping of exon 25. The annotation of human L1CAM exon 25 refers to RefSeq transcript NM_00425 and is consistent with the previous literature (Mikulak et al., 2012). (B) Upper panel: schematic diagram of the human L1CAM genomic region containing the AS exon 25 (grey box). Black boxes = constitutive exons; thin lines = introns. Red and blue lines indicate the two possible AS reactions, and the two resulting isoforms are shown on the right. Lower panel: RT-PCR analysis of AS of the human L1CAM exon 25 in different human tissues and in two EC lines (hCMEC/D3 and HUVEC). (C) Scheme of AS events of the mouse L1cam exon 25 and RT-PCR analysis in mouse tissues, ECs freshly purified from mouse lung and mouse EC lines (moEC, EmbEC, luEC and lu2EC). Asterisk indicates an additional band (more evident in moEC and EmbEC) corresponding to a novel transcript deleted of exons 25, 26 and 27.

https://doi.org/10.7554/eLife.44305.003

Alternative splicing generates a novel soluble form of L1CAM

Skipping of exon 25 results in an in-frame deletion of a 44-amino acid sequence (45 in mouse) that encompasses the entire transmembrane (TM) domain of L1CAM (Figure 2A). This suggests that AS of exon 25 could affect L1CAM localization and, hence, its activity. To test this hypothesis, we selected an immortalized endothelial cell line of murine origin, moEC (Lampugnani et al., 2002; Taddei et al., 2008), because it expresses no or very little endogenous L1CAM (Figure 2B) and, therefore, is amenable to gain-of-function studies. As shown in Figure 1—figure supplement 1, ECs express the non-neural isoform of L1CAM mRNA, which lacks exons 2 and 27 (Schäfer and Altevogt, 2010). Thus, exon 25 was deleted from non-neural mouse L1cam in order to obtain L1-ΔTM. MoEC were stably transduced with either full-length, non-neural L1CAM (L1-FL) or with L1-ΔTM. Immunoblotting of total extracts from L1-FL-expressing cells showed the expected doublet pattern (Figure 2B), with the upper band representing the mature, fully glycosylated cell-surface form, and the lower band corresponding to the precursor form (Moulding et al., 2000; Zisch et al., 1997). In contrast, L1-ΔTM-expressing moEC showed a single band that migrated slightly faster than the lower band of L1-FL (Figure 2B), consistent with the deletion of exon 25. The lack of the TM domain might affect the subcellular localization of L1CAM. To address this question, we performed immunofluorescence staining for L1CAM on moEC expressing the two isoforms. Only L1-FL was prominently exposed on the cell surface of moEC, while cells expressing L1-ΔTM exhibited mainly a cytoplasmic localization (Figure 2C). Based on the deletion of the TM domain and on the absence of membrane staining, we hypothesized that L1-ΔTM is released into the extracellular space and represents a novel soluble form of the protein. Indeed, conditioned medium (CM) from L1-ΔTM-expressing moEC contained high amounts of L1-ΔTM (Figure 2D). Of note, the protein in the CM retained the cytoplasmic tail (Figure 2—figure supplement 1). This confirmed the release of the entire molecule as opposed to the proteolytic cleavage of full-length L1CAM, which results in the shedding of its extracellular portion (Figure 2D; Mechtersheimer et al., 2001). Both the cytoplasmic localization of L1-ΔTM and its release into the CM were also confirmed in luEC, another murine EC line (Figure 2—figure supplement 1).

Figure 2 with 1 supplement see all
Expression, cell surface localization and release of L1CAM isoforms.

(A) The amino acid sequence of the mouse and human L1CAM region across the membrane. The transmembrane domain (blue rectangle), with 91% identity between mouse and human, and the sequence encoded by exons 25 (grey rectangle) are indicated. Bottom: schematic structure of L1CAM, showing the six Ig domains (Ig) and the five FN type-III repeats (Fn) in the extracellular portion. (B) Immunoblotting for L1CAM on lysates from moEC stably over-expressing the L1CAM isoforms (L1-FL or L1-ΔTM) or the empty vector (Vector). Immunoblotting for α-Tubulin served as loading control. (C) Representative images from the immunofluorescence analysis of L1CAM (red) and the endothelial cell surface marker CD31 (green) on moEC overexpressing either L1-FL or L1-ΔTM (confocal sections, z axis; scale bar 10 μm). Arrowheads show L1-FL localization at the cell surface, while arrows show the cytosolic localization of L1-ΔTM. The graph (right panel) shows the quantitation of the cellular localization of the two L1CAM isoforms. Values represent means ±SD from five different fields in each condition. Comparisons between experimental groups were done with two-sided Student’s t-test; ***p<0.001. (D) Immunoblotting for L1CAM on lysates (Lys) and conditioned media (CM) from moEC stably overexpressing either L1-FL or L1-ΔTM. Equal amounts of protein extracts and volumes of CM derived from equal numbers of producing cells (see Materials and methods) were analysed.

https://doi.org/10.7554/eLife.44305.005

Interestingly, L1-ΔTM was detected in the culture medium as a single band that migrated slower than the protein found in the cell lysate (Figure 2D). We hypothesized that the different size was accounted for by the glycosylation state of the protein. This was confirmed by the forced expression of L1-ΔTM in N-acetylglucosaminyltransferase I-deficient HEK293 cells [GnTI(-)] (Reeves et al., 2002). The latter, indeed, released a form of L1-ΔTM that exhibited a lower molecular weight as compared to wild-type HEK293 cells (Figure 2—figure supplement 1). These findings support the notion that L1-ΔTM is released in its mature, fully glycosylated form.

In order to confirm the release of endothelial L1-ΔTM in an endogenous system, we employed lu2EC, an immortalized mouse endothelial cell line that expresses relatively high levels of L1cam (Figure 2—figure supplement 1). These cells also express endogenous L1-ΔTM (Figure 2—figure supplement 1). When the lu2EC-derived CM was immunoblotted with the antibody against the cytoplasmic tail of L1CAM, we found high levels of L1-ΔTM (Figure 2—figure supplement 1), confirming its release into the extracellular space. To compare our findings in ECs with a non-endothelial cell type, we used the mouse melanoma cell line B16, which also expresses high levels of endogenous L1CAM (Linnemann and Bock, 1986; Magrini et al., 2014), but does not express L1-ΔTM (Figure 2—figure supplement 1). While we could detect high levels of cell-associated L1CAM, no C-terminus-containing L1CAM was detected in the CM of B16 cells (Figure 2—figure supplement 1), supporting the hypothesis that the AS of L1CAM results in the release of L1-ΔTM in ECs.

Collectively, our results indicate that skipping of L1CAM exon 25 generates a novel isoform of the protein that is released in the extracellular compartment.

L1-ΔTM regulates endothelial cell function

To investigate the biological role of the L1-ΔTM isoform in ECs, we focused on their ability to form capillary-like tubes in three-dimensional matrices, which reflects their angiogenic potential (Di Blasio et al., 2014). Therefore, we assayed control and L1-ΔTM-expressing ECs for tube formation on Matrigel. As shown in Figure 3—figure supplement 1, Figure 3A and Video 1, L1-ΔTM enhanced significantly the tube forming ability of moEC, thus suggesting that it is endowed with angiogenic properties. The direct role of L1-ΔTM in moEC tube formation was probed with 324, a L1CAM-neutralizing antibody (Appel et al., 1993; Di Sciullo et al., 1998). As shown in Figure 3B, the antibody 324, but not a control antibody, abolished the tube-forming potential of moEC. The results of this proof-of-concept experiment also support the neutralization of vascular L1-ΔTM as a potential strategy to interfere with the angiogenic process.

Figure 3 with 1 supplement see all
L1-ΔTM stimulates angiogenesis in an autocrine/paracrine fashion.

(A) Quantitation of tube formation by transduced moEC. (B) Transduced moEC were subjected to tube formation assays in the presence of either anti-L1CAM clone 324 or a control, irrelevant antibody (IgG). (C) Quantitation of tube formation by parental moEC treated with CM from moEC transduced either with the empty vector (Vector) or with L1-ΔTM. (D) Quantitation of tube formation assays on moEC untreated or treated with increasing concentrations of recombinant L1-ΔTM. (E) Schematic illustration of the mechanism of action of the morpholino oligonucleotide (L1–SB), which binds to the exon 25/intron 25 junction of L1cam, thus preventing the recruitment of the spliceosome and, hence, impairing the inclusion of exon 25. (F) lu2EC transfected with either an irrelevant morpholino (Ctr) or with L1-SB were analyzed by RT-PCR for the AS of L1cam exon 25 (left, top panel), whereas CM from the same cells were analyzed in immunoblotting with the L1CAM antibody (left, bottom panel). Parental moEC were subjected to tube formation assays in the presence of CM from Ctr- or L1-SB-transfected lu2EC (right panel). (G) Representative images and quantitation of vessel density in matrigel plugs pre-mixed with the CM from ECs transduced with either the empty vector (Vector), L1-FL or L1-ΔTM, and then implanted subcutaneously into C57Bl/6 mice (n = 3 mice/group). Matrigel plugs containing FGF2 served as positive control. Scale bar, 100 μm. Right panel: CD31+ vessels were counted in five different fields. (H) Left panels: immunoblots for phospho-FGFR1 (pFGFR1) and total FGFR1 (FGFR1) on serum-starved moEC left untreated or treated with recombinant L1-ΔTM (20 μg/ml) for 10 or 30 min. The blots were obtained from the same gel, the white line between the blots indicates the removal of intervening lanes. Right panel: FGFR1 phosphorylation in three biological replicates was quantitated by calculating the ratio between phospho-FGFR1 and total FGFR1. Data are normalized against the basal phosphorylation in untreated cells (indicated by the red dashed line). (I) moEC transduced with the empty vector (Vector) or with L1-ΔTM were subjected to tube formation assays in the presence of either the FGFR1 inhibitor PD173074 (PD) or DMSO as a control. For each analysis, data are expressed as means ± SEM from three independent experiments. Comparisons between experimental groups were done with two-sided Student’s t-tests; **p<0.01, ***p<0.001.

https://doi.org/10.7554/eLife.44305.007
Video 1
L1-ΔTM promotes EC tube formation.

Time-lapse videomicroscopy of tube formation on moEC transduced either with the empty vector (A) or with L1-ΔTM (B).

https://doi.org/10.7554/eLife.44305.009

Based on the substantial release of L1-ΔTM into the extracellular space, we asked whether the molecule could also exert its biological function as a soluble factor in a paracrine fashion. To address this question, parental moEC were subjected to tube formation assays in the presence of the CM from moEC expressing either L1-ΔTM or the control vector. ECs exposed to the CM from L1-ΔTM-expressing cells exhibited higher tube-forming activity than those exposed to control medium (Figure 3C) or to the CM from L1-FL-expressing cells (not shown). Similar results were obtained by using CM from luEC expressing either L1-ΔTM or the control vector (Figure 3—figure supplement 1). To further verify the angiogenic activity of soluble L1-ΔTM, we treated parental moEC with a purified, recombinant version of the protein produced in mammalian cells (Figure 3—figure supplement 1). Indeed, recombinant soluble L1-ΔTM induced moEC tube formation in a dose-dependent manner (Figure 3D), thus confirming its ability to stimulate EC remodeling and morphogenesis. In order to validate our results in an EC model with endogenous L1-ΔTM, we treated lu2EC with a morpholino oligonucleotide that selectively prevents the inclusion of L1cam exon 25 (Figure 3E). As shown in Figure 3F, this resulted in increased expression and extracellular release of endogenous L1-ΔTM. Importantly, lu2EC exposed to the CM from morpholino-treated cells exhibited higher tube-forming activity than those exposed to control CM, thus confirming the functionality of endogenous L1-ΔTM (Figure 3F).

Finally, we aimed at validating our findings in an in vivo assay of angiogenesis. Mice underwent subcutaneous implantation of Matrigel plugs containing CM from either L1-ΔTM- or L1-FL-expressing ECs or from control cells. Neovascularization was markedly induced by L1-ΔTM-containing CM, while a weaker effect was observed with the CM from L1-FL-expressing cells (Figure 3G). This strongly supports the angiogenic function of L1-ΔTM.

FGFR1 signaling is required for L1-ΔTM-induced tube formation

Previous studies implicated fibroblast growth factor receptor (FGFR) signaling as an effector of L1CAM in different cellular contexts (Díaz-Balzac et al., 2015; Kulahin et al., 2008; Mohanan et al., 2013; Williams et al., 1994; Zecchini et al., 2008). However, the L1CAM/FGFR interplay in ECs has not been investigated. Given the well-characterized role of FGFR function in vascular biology and angiogenesis (Ronca et al., 2015), we hypothesized that the pro-angiogenic effect of L1-ΔTM was mediated by FGFR. Among the four FGFR family members, moEC express only FGFR1 (data not shown) (Giampietro et al., 2012), as previously reported for other EC types (Giacomini et al., 2016; Javerzat et al., 2002). To determine if L1-ΔTM function could be mediated by FGFR1, we first investigated whether soluble L1-ΔTM affects FGFR1 activation. As shown in Figure 3H, treating parental moEC with recombinant L1-ΔTM resulted in increased phospho-FGFR1, consistent with the L1-ΔTM-induced activation of FGFR1 signaling. Moreover, when L1-ΔTM-expressing moEC were subjected to tube formation assay in the presence of the small-molecule FGFR1 inhibitor PD173074 (Skaper et al., 2000), L1-ΔTM-dependent tube-forming activity was reduced to the level of control cells (Figure 3I). Thus, our data implicate FGFR1 signaling as an effector of L1-ΔTM in ECs.

NOVA2 controls alternative splicing of L1-ΔTM in ECs

To gain further insights into the molecular mechanisms regulating the AS of L1cam in endothelium, we analyzed the sequence of mouse L1cam exon 25 and its flanking intronic regions, using SFmap (http://sfmap.technion.ac.il/) (Paz et al., 2010; Akerman et al., 2009) to search for putative binding sites of RNA-binding proteins. We sorted the results based on: i) the predicted ability of the RNA-binding protein to promote exon 25 skipping; ii) the presence of clusters of putative binding sites for a given RNA-binding protein, which are expected to enhance binding affinity; iii) the evolutionary conservation of the identified motifs; iv) the known expression of the identified factor in ECs. This analysis resulted in the identification of clustered and evolutionarily conserved putative binding sites for NOVA2, hnRNP A1 and SRSF3 (Figure 4—figure supplement 1), three factors previously reported to be expressed in ECs (Giampietro et al., 2015; Holly et al., 2013; Lomnytska et al., 2004).

To investigate the role of the identified candidate splicing factors in the AS of L1cam, we first performed a splicing assay in HeLa cells co-transfected with a minigene (p-L1) encompassing exons 24, 25, and 26 of L1cam along with the flanking intron sequences (Figure 4A) and the candidate splicing factors or with the empty vector. As shown in Figure 4B and Figure 4—figure supplement 1, skipping of L1cam exon 25 in the minigene was only observed upon overexpression of NOVA2, a key regulator of AS in ECs (Giampietro et al., 2015). In contrast, the overexpression of hnRNP A1 and SRSF3 had no effect on the skipping of exon 25, suggesting that the latter is a NOVA2-specific effect. To support a direct and specific role of NOVA2 in controlling L1cam AS, we mutated YCAY (Y = C/U) motifs, which represent putative binding sites for NOVA proteins (Ule et al., 2006), in L1cam exon 25 to ACAY, a sequence that reduces NOVA2 binding (Jensen et al., 2000). We found that mutations in only three repeats (Mut3) had a limited effect, whereas mutations in five repeats (Mut5) decreased skipping of L1cam exon 25 caused by NOVA2 overexpression (Figure 4C). These results are consistent with the dose-dependent binding of NOVA2 to its pre-mRNA targets (Darnell, 2006; Leggere et al., 2016) and further supported the involvement of NOVA2 in the AS regulation of L1cam exon 25. Such a hypothesis was also sustained by the following observations: i) the higher expression of NOVA2 in freshly purified ECs from mouse lung and in lu2EC as compared with total mouse lung or melanoma cell line B16, respectively, was accompanied by the skipping of L1cam exon 25 (Figure 4—figure supplement 2 and Figure 2—figure supplement 1); and ii) L1cam exon 25 emerged as a novel NOVA2 target in ECs from the RNA-seq data in NOVA2-knockdown ECs (Giampietro et al., 2015) (Supplementary file 2) (see Materials and methods).

Figure 4 with 3 supplements see all
L1-ΔTM production is regulated by NOVA2 in ECs.

(A) The mouse L1cam cassette, encompassing exons 24, 25 and 26, with the intervening intronic sequence was used to generate the p-L1 wild-type (WT) minigene. Mutated minigenes (Mut3 and Mut5) were generated by mutations in either three or all five NOVA-binding sites in exon 25 (YCAY repeats were mutated to ACAY). Boxes = exons; thin lines = introns; pCMV = promoter; BGHpA = polyadenylation sequence; red arrows indicate the annealing positions of the primers used for RT-PCR. (B) AS of transcripts of the WT minigene upon co-transfection of HeLa cells with either HA-NOVA2, T7-hnRNP A1 (T7–A1), or the empty vector. The ectopic expression of NOVA2 and hnRNP A1 was confirmed by western blotting with anti-HA and anti-T7 antibodies, respectively. (C) AS of transcripts from the WT and mutated minigenes in co-transfected HeLa cells. The histogram shows the ratio between skipping and inclusion of L1cam exon 25. Data indicate means ± SEM calculated from five independent experiments (n = 5). Tukey’s multiple comparisons or two-sided Student’s t-test were used for comparisons between experimental groups; *p<0.05; **p<0.01. (D) L1cam mouse genomic region comprising the AS exon 25 (grey box). Black boxes = constitutive exons; thin lines = introns; blue dot = YCAY cluster within exon 25 predicted to function as NOVA2-binding site. Bottom diagrams illustrate the inclusion (left) or the NOVA2-induced skipping of exon 25 (right). (E) AS of mouse L1cam exon 25 as determined by RT-PCR in moEC stably overexpressing HA-tagged NOVA2 cDNA. (F) AS of mouse L1cam exon 25 in moEC transduced with an shRNA against Nova2 or with a control shRNA (Ctr). The percentage of exon inclusion was calculated as described in Materials and methods and is shown below the gels. (G) CLIP was performed in moEC with anti-NOVA2 or control IgG. NOVA2-bound RNA was analyzed by RT-qPCR with L1cam primers E25 (annealing to the YCAY cluster), E2 (annealing to the exon 2), I2 (annealing to the intron 2), E26 (annealing to exon 26), I26 (annealing to the intron 26) and I27 (annealing to the intron 27 and exon 28). Binding of NOVA2 was calculated as % of input (see Materials and methods). Black arrows in the top diagram show the annealing position of the three primer sets. Data are expressed as means ± SEM calculated from three independent experiments (n = 3). ***p<0.001.

https://doi.org/10.7554/eLife.44305.010

To investigate the causal relationship between NOVA2 expression and AS of the endogenous L1cam, we performed gain- and loss-of function studies in moEC (Figure 4D–F and Figure 4—figure supplement 3). In particular, forced expression of NOVA2 increased skipping of L1cam exon 25 (Figure 4E and Figure 4—figure supplement 3). Conversely, in NOVA2-depleted moEC (Figure 4—figure supplement 3) the skipping of L1cam exon 25 was markedly reduced (Figure 4F). The NOVA2-mediated AS regulation of L1cam exon 25 was also confirmed in lu2EC, another murine EC line (Figure 4—figure supplement 3).

Whether NOVA2 promotes exon skipping or inclusion depends on the location of its binding sites (i.e. YCAY clusters) in the pre-mRNA targets (Ule et al., 2003). In particular, NOVA2 usually induces exon skipping when bound to the exonic or upstream intronic region, while it stimulates exon inclusion when interacting with downstream intronic region. In the case of L1cam exon 25, the YCAY repeats are located within exon 25 (Figure 4A), consistent with the NOVA2-induced exon skipping observed in mouse ECs. Notably, the YCAY cluster is conserved between mouse and human L1CAM exons 25 with six repeats present in the human sequence (Figure 4—figure supplement 3). Accordingly, NOVA2 overexpression promotes skipping of L1CAM exon 25 also in human ECs (Figure 4—figure supplement 3).

To determine if NOVA2 directly regulates AS of the endogenous L1cam, we carried out UV crosslinking and immunoprecipitation (CLIP), which allows to identify direct protein-RNA interactions in live cells (Ule et al., 2006). RNA from UV cross-linked ECs was immunoprecipitated by using anti-NOVA2 or control antibodies and then analyzed by RT-qPCR with primers spanning the YCAY cluster within L1cam exon 25. Primers that span either exon 26 or intron 26 were used as negative controls (Figure 4G). As shown in Figure 4G, NOVA2 bound to the endogenous L1cam transcript at the level of exon 25, while we observed no binding with either exon or intron 26. These data indicated a direct and specific interaction of NOVA2 with L1cam exon 25.

NOVA2 has been implicated also in the inclusion of exons 2 and 27 in neural cells (Mikulak et al., 2012). However, both RT-PCR and CLIP data showed no involvement of NOVA2 in the AS of these two exons in ECs (Figure 4G and Figure 1—figure supplement 1), further supporting the specific effect of NOVA2 on exon 25 in this cell type.

Collectively, our results support the notion that NOVA2 promotes skipping of L1cam exon 25 by binding to the YCAY motifs located within this exon.

Clinical relevance of NOVA2-mediated AS of L1CAM in ovarian cancer vessels

We have recently described the expression of NOVA2 in vascular endothelium (Giampietro et al., 2015). Furthermore, our earlier reports demonstrated that L1CAM is expressed in tumor-associated vasculature (Maddaluno et al., 2009; Magrini et al., 2014). Taken together with the data presented here, these findings raise the hypothesis that NOVA2 regulates AS of L1CAM in cancer vessels. To test this possibility, we selected human ovarian carcinoma (OC) as a suitable model system. In fact, we found a markedly higher number of NOVA2-positive vessels in OC (identified via staining with the endothelial marker CD31) than in healthy ovaries (Figure 5A and Figure 5—figure supplement 1). The abundance and the vessel-restricted expression of NOVA2 in OC were also confirmed in tissue samples from the Human Protein Atlas project (https://www.proteinatlas.org/) (Uhlén et al., 2015) (Figure 5—figure supplement 1). The percentage of L1CAM-positive vessels was also dramatically increased in OC samples as compared to normal ovary (Figure 5A). In addition, NOVA2 was often co-expressed with L1CAM in OC vessels (Figure 5B and Figure 5—figure supplement 2). Thus, we applied RT-PCR to examine the AS of L1CAM in ECs isolated from OC (HOC-EC). As shown in Figure 5C, the L1-ΔTM isoform was readily detected in HOC-EC from seven independent OC samples. To test whether vascular L1-ΔTM in OC is associated with tumor angiogenesis, we measured the vessel density in a small cohort of OC samples pre-classified as L1-ΔTM-positive or negative by RT-qPCR (Figure 5—figure supplement 2). A significantly higher vessel density was found in L1-ΔTM-positive tumors (Figure 5—figure supplement 2). Furthermore, among the tumors which exhibited L1-ΔTM expression, the levels of L1-ΔTM correlated with vessel density (r = 0.7671; p<0.01), measured by CD31 immunostaining (Figure 5D). These findings imply that the AS of L1CAM correlates with the degree of OC vascularization, which is consistent with a proangiogenic function of L1-ΔTM in this tumor type. To further assess the clinical relevance of our findings, we investigated the prognostic value of NOVA2 in OC, profiting from the RNA sequencing analysis of 372 OC patients performed through The Cancer Genome Atlas (TCGA) program. As shown in Figure 5E, higher expression of NOVA2 correlated with shorter overall survival of the patients (HR: 1.486; p=0.003). Taken together, these results suggest that NOVA2 promotes AS of the L1CAM pre-mRNA in OC vessels, thus accounting for the vascular expression of L1-ΔTM, and highlight the proangiogenic role and the prognostic value of the NOVA2/L1-ΔTM axis in OC.

Figure 5 with 2 supplements see all
L1CAM and NOVA2 are co-expressed in ovarian cancer vessels.

(A) Quantification of NOVA2- and L1CAM-positive vessels in healthy ovaries (n = 5) and high-grade serous ovarian cancer (OC) (n = 5). Data are expressed as the percentage of vessels positive for either NOVA2 (left) or L1CAM (right) over the total of CD31-positive vessels, and are shown as means ± SEM. Comparisons between experimental groups were done with two-sided Student’s t-test; ***p<0.001. (B) Serial sections of OC samples were stained for L1CAM (brown) or NOVA2 (green). Arrows indicate L1CAM-positive vessels (panels a and b), and arrowheads indicate NOVA2 staining in EC nuclei (panels c and d). The right panels (b, d) show higher magnification of the areas delimited by dashed boxes. Scale bars: 100 μm, panels a and c; 50 μm, panels b and d. (C) AS of the human L1CAM exon 25 in seven EC batches purified and cultured from human ovarian carcinoma (HOC-ECs). (D) The skipping of L1CAM exon 25 was measured in OC samples (n = 13) as the ratio between L1-ΔTM and total L1CAM (determined by gel quantitation of RT-PCR products; see Figure 5—figure supplement 2). Microvessel density was measured in the same samples by CD31 staining and correlated with the AS of L1CAM exon 25. (E) Kaplan-Meier plot of overall survival in OC patients classified according to NOVA2 expression (red curve, high expression; blue curve, low expression).

https://doi.org/10.7554/eLife.44305.014

Discussion

Our data implicated for the first time the splicing factor NOVA2 in the generation of a novel, EC-specific isoform of the cell adhesion molecule L1CAM, referred to as L1-ΔTM. Due to NOVA2-induced skipping of exon 25 that encodes the TM domain, L1-ΔTM is no longer associated to the cell surface and, hence, is released in the extracellular space. Consistent with the expression of NOVA2 in vascular ECs (Giampietro et al., 2015), the latter express and release high levels of L1-ΔTM.

We demonstrated that L1-ΔTM increases the ability of ECs to form tube-like structures in vitro and stimulates neovascularization in vivo. These data point to L1-ΔTM as a bona fide angiogenic factor which, however, belongs to a class of molecules highly divergent from the classic polypeptide growth factors that exert this function (vascular endothelial growth factors, fibroblast growth factors, etc.). To our knowledge, L1-ΔTM provides the first example of an immunoglobulin-like cell adhesion molecule converted into a soluble angiogenic factor through AS removal of the exonic sequence that encodes the TM domain. AS of the TM domain occurs in a broad spectrum of human genes (Xing et al., 2003), yet for most of them the biological significance and the functional outcome of such an event remain elusive. Based on our findings, it is conceivable that this post-transcriptional modification expands the repertoire of biologically active proteins that are released into the extracellular compartment.

We discovered that ECs acquire angiogenic properties not only upon ectopic expression of L1-ΔTM, but also when exposed to exogenously added L1-ΔTM (either as CM or as a recombinant protein). This supports the notion that extracellular L1-ΔTM can promote angiogenesis via both autocrine and paracrine stimulation. The latter mode of action, in particular, might represent a way to amplify the angiogenic signal provided by vascular L1CAM. Indeed, in most cases only a fraction of vessels within tumor and inflammatory tissues (i.e. the most prominent conditions where vascular L1CAM is detected), exhibits L1CAM expression (Issa et al., 2009; Kaifi et al., 2006; Maddaluno et al., 2009; Magrini et al., 2014). Thus, the AS-mediated generation of L1-ΔTM, and hence its release, would contribute to spread the proangiogenic stimulus also to L1CAM-negative vessels.

In different cellular contexts, such as neurons, L1CAM frequently engages in homophilic L1CAM-L1CAM interactions between adjacent cells, which underlie its function in cell-cell adhesion (Maness and Schachner, 2007). However, our experiments were conducted in two EC lines, moEC and luEC, that express no or very little endogenous L1CAM. This implies that, regardless of autocrine or paracrine activity, L1-ΔTM stimulates ECs by heterophilic interactions with different surface molecules. Accordingly, the extracellular portion of L1CAM has been reported to interact with a wide spectrum of membrane proteins, including different integrins, CD24, NCAM, F11R, neuropilins, etc. (Haspel and Grumet, 2003). With regard to potential L1-ΔTM interactors, one of the most intriguing aspects of our study is related to the crosstalk between L1-ΔTM and FGFR1. Following the pioneering studies of Doherty and Walsh in neuronal systems (Williams et al., 1994), L1CAM has been proposed to interact with the FGFR signaling machinery in different experimental settings (Mohanan et al., 2013; Son et al., 2011; Zecchini et al., 2008), possibly entailing a direct binding between the two molecules (Kulahin et al., 2008). Our data show for the first time a functional interplay between L1-ΔTM and FGFR1 in ECs, and implicate it in L1-ΔTM-induced angiogenesis. Future studies should elucidate whether interfering with the L1-ΔTM/FGFR1 crosstalk will open novel perspectives for antiangiogenic therapy.

In neural cell types, NOVA2 is required for the inclusion of the L1CAM exons 2 and 27 (Mikulak et al., 2012). This is not the case in ECs, where even upon ectopic over-expression of NOVA2 exons 2 and 27 are not included in the L1CAM mRNA (Figure 1—figure supplement 1). Moreover, despite the high expression of NOVA2 in the nervous system (Yano et al., 2010; Giampietro et al., 2015), we did not detect L1-ΔTM in human or mouse brain (Figure 1), which implies that NOVA2 is not involved in the AS of exon 25 in neural cells. Interestingly, our CLIP data on ECs revealed the binding of NOVA2 to exon 25, while no binding was detected with the regions flanking the neural-specific exons 2 and 27 (Figure 4G). Overall, these observations point to differential and cell type-specific interactions of NOVA2 with the L1CAM pre-mRNA that, in turn, underlie distinct AS outcomes. Future studies should aim at identifying the molecular determinants of such a cell type specificity.

NOVA2 is significantly up-regulated in OC vasculature as compared to vessels in healthy ovary. Of note, NOVA2 expression is detected only in the vascular endothelium of OC and, in particular, in the nucleus of ECs. It is remarkable that, in spite of such a restricted expression pattern, NOVA2 has a prognostic value in OC, given its association with shorter patients’ survival. To the best of our knowledge, NOVA2 is the only SRF reported to be upregulated in cancer vasculature, including OC (this study) and colorectal carcinoma (Gallo et al., 2018). This implies that NOVA2-mediated AS might play a relevant, yet underappreciated, role in the phenotypic and functional aberrancies of the tumor vessels (Carmeliet and Jain, 2011). Consistent with the proposed role of the NOVA2/L1-ΔTM axis in tumor angiogenesis, increased NOVA2 levels are frequently accompanied by the expression of L1-ΔTM in tumor vessels. Furthermore, L1-ΔTM expression correlates with OC vascularization, although further studies should investigate the causal role of L1-ΔTM in tumor angiogenesis. These data, together with our observations on the role of vascular L1CAM in tumor angiogenesis and progression (Magrini et al., 2014) and on the proangiogenic activity of L1-ΔTM, point to NOVA2 as a potential driver of OC neovascularization. In addition, our results further strengthen the rationale for testing vascular L1CAM as a novel target for antiangiogenic strategies. Indeed, interfering with the function of cell surface-associated endothelial L1CAM (Magrini et al., 2014) and, at the same time, with that of extracellular L1-ΔTM might result in efficient repression of cancer angiogenesis.

The translational implications of our findings extend beyond the therapeutic area. Based on its specific up-regulation in OC vessels (and likely in other cancer types as well), we speculate that L1-ΔTM could serve as a new circulating biomarker of cancer-associated vasculature. In this case, it may become necessary to discriminate between vessel-derived L1-ΔTM and the ectodomain of transmembrane L1CAM which is released by various cell types, including tumor cells, upon proteolytic cleavage (Kiefel et al., 2012; Yu et al., 2016). Assays aimed at detecting the cytosolic domain of circulating L1CAM would serve this purpose. Circulating L1-ΔTM could be harnessed for diagnostic and prognostic purposes, ranging from the detection of primary and recurrent OC to monitoring the therapeutic response, all objectives that would improve dramatically the clinical management of OC patients. Along this line, the circulating levels of vessel-derived L1-ΔTM could offer a novel predictive tool to identify patients eligible for antiangiogenic therapies.

In summary, we provide evidence that L1-ΔTM is a novel, soluble isoform of L1CAM generated in ECs through NOVA2-mediated AS. L1-ΔTM exhibits proangiogenic activity and is up-regulated in tumor vessels, which may have high translational and clinical relevance.

Materials and methods

Cell culture

Human cervix carcinoma HeLa cells (ATCC, CCL-2) were grown in Dulbecco’s modified Eagle’s medium (DMEM, Euroclone) supplemented with 10% fetal bovine serum (FBS, Euroclone) and 2 mM L-glutamine (Lonza). Human embryonic kidney (HEK) 293 cells (FreeStyle 293 F Cells, Gibco) were grown in FreeStyle 293 Expression Medium (Gibco). N-acetylglucosaminyltransferase I-deficient HEK293 cells [GnTI(-)] (Reeves et al., 2002) were grown in FreeStyle 293 Expression Medium supplemented with 0.1% Pluronic F-127 (Sigma-Aldrich) and 1% FBS.

Vascular endothelial (VE) cadherin-positive ECs, here referred to as moEC, have been described in Lampugnani et al. (2002) and Taddei et al. (2008). An extensive characterization confirmed the endothelial nature of these cells (Lampugnani et al., 2003; Taddei et al., 2008). Mouse lung-derived luEC and lu2EC were described in Magrini et al. (2014), and Bazzoni et al. (2005), respectively. Primary EC were isolated from mouse lung with anti-CD31 immunomagnetic beads as described (Malinverno et al., 2017). More than 99% isolated cells were positive for VE-cadherin and CD31 by immunofluorescence (Figure 4—figure supplement 2). Furthermore, isolated cells exhibited a marked enrichment in the mRNA for VE-cadherin and CD31, while no or very little expression was detected for epithelial (E)-cadherin and alpha-smooth muscle actin (Figure 4—figure supplement 2).

Mouse EC lines derived from whole embryo (EmbEC), lu2EC and moEC were cultured in DMEM-High Glucose (Lonza) with 10% FBS, 2 mM L-glutamine, 100 U/L penicillin/streptomycin (Sigma-Aldrich), 1 mM sodium pyruvate (Sigma-Aldrich), 25 mM HEPES (Sigma-Aldrich), 100 μg/ml heparin (from porcine intestinal mucosa; Sigma-Aldrich) and 50 μg/ml EC growth supplement (ECGS from bovine pituitary gland; Sigma-Aldrich). LuEC were cultured in MCDB131 medium (Sigma-Aldrich) supplemented with 20% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate (Gibco), 100 μg/ml heparin and 50 μg/ml ECGS as previously described (Magrini et al., 2014). To enhance EC adhesion, plates were coated with 0.1% porcine gelatin (Difco) and incubated overnight at 37°C before seeding. Human umbilical vein EC (HUVEC, isolated as in (Giampietro et al., 2015) and HUVEC/TERT2 (Evercyte) were cultured on porcine gelatin-coated plates in MCDB131 containing 20% FBS and the same supplements as moEC and luEC.

Human ovarian cancer-derived EC (HOC-EC) have been described previously. HOC-EC cultures were found to contain at least 99% endothelial marker-positive (LDL uptake, CD31, von Willebrand Factor) cells, and <1% alpha-smooth muscle actin-positive cells (Ghilardi et al., 2015).

Human immortalized cerebral microvascular ECs (hCMEC/D3) were provided by PO Couraud and cultured as described previously (Weksler et al., 2013). hCMEC/D3 cells were grown on Collagen type I coated dishes (Corning).

Mouse melanoma B16F10 cells (hereafter referred to as B16) were purchased from ATCC and cultured in DMEM, 10% FBS.

All cells were routinely tested for mycoplasma with a PCR-based method. Whenever applicable, cell line authentication was performed with the GenePrint STR System typing kit (Promega).

Plasmids

The cDNAs encoding HA-tagged, full-length mouse L1cam (deleted of the neural-specific exons 2 and 27) and its TM—deleted L1cam isoform (deleted of exons 2, 25 and 27) (Figure 1—figure supplement 1) were generated by PCR-mediated mutagenesis of pcDNA3.1-Hygro/L1 construct (Magrini et al., 2014) and then cloned into the pcDNA3.1-Hygro(-) (Invitrogen) or in the pLenti-III-HA (ABM Inc) by using standard DNA cloning procedures. To generate the p-L1 WT minigene (Figure 4), the genomic mouse L1cam cassette was amplified with primers p-L1_F/R and then cloned into the EcoRI and XbaI restriction sites of pcDNA3.1(+) (Invitrogen). In p-L1 Mut minigenes (Mut3 and Mut5), the NOVA2-binding sites TCAT were replaced with ACAT by PCR-mediated mutagenesis of p-L1 WT minigene. HA-tagged human NOVA2 cDNA was PCR amplified with primers pcDNA-NOVA2_F/R and cloned into the BamH1 and XhoI restriction sites of the pcDNA3.1(+), whereas T7-tagged hnRNP A1 expression vector was generated as described previously (Bonomi et al., 2013b). The expression vector for T7-SRSF3 was kindly provided by J. Caceres (MRC Human Genetics Unit, University of Edinburgh, UK).

To produce recombinant L1-ΔTM protein (see below), the L1-ΔTM cDNA was amplified with pUPE-L1_F/R primers from pcDNA Hygro-L1-ΔTM. The resulting amplicon, bearing in-frame 5'-BamHI and 3'-NotI restriction sites was then sub-cloned into pUPE.06.45 mammalian expression vector (U-Protein Express B.V., Utrecht, The Netherlands), bearing a cystatin signal peptide followed by N-terminal 6xHis, 3xStrepII tags, and a specific cleavage site for Tobacco Etch Virus protease (TEV) preceding the 5'-BamHI restriction site. All PCR products were verified by sequencing, whereas all primers are listed in Supplementary file 1.

Cell transfection

HeLa cells were transiently transfected with Lipofectamine 3000 (Invitrogen), according to the manufacturer’s protocol. luEC were stably transfected with pcDNA-Hygro/L1-FL or pcDNA-Hygro/L1-ΔTM or the empty vector pcDNA-Hygro(-) (see below), followed by the selection of positive clones as previously described (Magrini et al., 2014). HEK293 and HEK293 GntI(-) cells were transfected using polyethyleneimine (Polysciences, Germany) as described (Durocher et al., 2000).

Lentivirus production and transduction

To obtain viruses expressing L1CAM isoforms, mouse L1cam cDNAs were cloned in the pLenti-III-HA vector (ABM Inc, see above). Lentiviruses were produced using HEK293T as recipient cells (ATCC, CRL-1573). Cells were co-transfected with 10 μg of each packaging vector pMD2.G (Addgene, plasmid #12259), pRSV-Rev (Addgene, plasmid #12253), pMDLg/pRRE (Addgene, plasmid #12251) and 10 μg of L1CAM-expressing lentiviral vectors by calcium phosphate precipitation method. After 24 hr, the medium containing the lentiviruses was filtered, supplemented with 8 μg/ml of polybrene (Sigma-Aldrich) and used to infect moEC.

MoEC were transduced with HA-NOVA2 or with shRNA vectors as described in (Giampietro et al., 2015), using lentiviral vectors carrying human HA-tagged NOVA2 cDNA (pLenti-GIII-CMVhumanNOVA2-HA, THP Medical Products) or shRNA for the mouse Nova2 gene (GIPZ shRNAs from Open Biosystems), respectively. After 48 hr of infection, the medium was refreshed and puromycin selection (3 μg/ml) was started. Since NOVA2 expression is regulated by EC density (Giampietro et al., 2015), for the analysis of L1CAM splicing NOVA2-knockdown moEC were used as confluent monolayers (500000 cells in 35 mm Petri dishes), whereas moEC overexpressing HA-tagged NOVA2 were tested at low density (500000 cells in 100 mm Petri dishes). hCMEC/D3 cells were transduced with lentiviral vectors carrying human HA-tagged NOVA2 cDNA (pLenti-GIII-CMVhumanNOVA2-HA, THP Medical Products) and after 48 hr, infected cells were selected with 3 μg/ml puromycin for 5 days.

RNAi

To ablate Nova2 in lu2EC, we used siRNA from Sigma-Aldrich (MISSION siRNA ID SASI_Mm01_00094763) and the corresponding negative control (MISSION siRNA Universal Negative Control #1). Transfection was performed with Lipofectamine RNAiMax (Invitrogen) in accordance with the manufacturer’s instructions. Two subsequent transfections (with 24 hr intervals) were performed with 13 nM siRNA, and cells were collected 24 hr after the second transfection. To knock down NOVA2 in HUVEC/TERT, we used siRNA from Sigma-Aldrich (MISSION siRNA ID SASI_Hs01_00220812) and the corresponding negative control (MISSION siRNA Universal Negative Control #1). Transfection was performed with Lipofectamine RNAiMax (Invitrogen) following the manufacturer’s instructions. Two subsequent transfections (with 24 hr intervals) were performed with 30 nM siRNA, and cells were collected 48 hr after the second transfection.

Morpholino treatment

Subconfluent (80–85%) lu2EC cells were transfected with the MO-L1-SB oligonucleotide (5’- CCTGTACATTTTCTAGGTTACCTGA-3’; GENE-TOOLS) at 15 μM plus 8 μM Endo-Porter PEG system (GENE-TOOLS) according to the manufacturer’s instructions. An irrelevant morpholino oligonucleotide (Standard Control Oligo, GENE-TOOLS) was used as control. After 16 hr, lu2EC were washed three times with PBS and starved for 24 hr in the following medium: DMEM High Glucose, 2 mM L-glutamine, 100 U/l penicillin streptomycin, 1 mM sodium pyruvate, 25 mM HEPES, 0.5% FBS. Conditioned media (CM) were collected and centrifuged for 10 min to remove cell debris.

Immunoblot analysis

Total protein extraction and immunoblot were performed as previously described in (Magrini et al., 2014). Briefly, total proteins were extracted by solubilizing cells in Laemmli buffer (4% SDS, 16% glycerol, 40 mM Tris-HCl pH 6.8). To ensure an equal loading, lysates were quantified using Pierce BCA protein assay kit (Thermo Fisher Scientific) and 20 μg loaded on gel. The amount of EC-derived CM analyzed by immunoblotting was normalized against the number of producing cells. Lysates and CM were separated using SDS–PAGE and analyzed by western blotting. The following primary antibodies were used: anti-NOVA2 C-16 (1:200; Santa Cruz Biotechnology), anti-α-Tubulin (1:50,000; Sigma-Aldrich), anti-HA High Affinity (1:1000; Roche), anti-T7 tag (1:5000; Novagen), anti-Vinculin (1:5000 Millipore), anti-L1CAM [1 μg/ml; (Magrini et al., 2014)], anti-L1CAM cytoplasmic domain [1:8000; Ral1cd, kindly provided by V Lemmon; (Schaefer et al., 2002)]; anti-FGFR1 (1:200; clone M2F12, Santa Cruz Biotechnology) and anti-phospho-FGFR1(Y653/654) (1:1000 Santa Cruz Biotechnology). The following secondary antibodies conjugated to horseradish peroxidase (Jackson ImmunoResearch) were used: anti-Mouse (1:5000), anti-Goat (1:5000), anti-Rat (1:5000) and anti-Rabbit (1:10000). Immunostained bands were detected using the chemiluminescent method (Clarity ECL Western Blotting Substrate, Bio-Rad) and images were obtained by ChemiDoc Imaging Systems (Bio-Rad).

Immunofluorescence

ECs were grown on gelatin-coated coverslips and then fixed with 4% PFA for 10 min at room temperature (RT). Membrane permeabilization was obtained incubating coverslips in ice-cold PBS and 0.5% Triton X-100 for 3 min at 4°C. Cells were then incubated for 1 hr at RT in a humid chamber with blocking solution (PBS, 2% BSA, 5% donkey serum, and 0.05% Triton X-100). Samples were then incubated for 2 hr with primary antibodies (anti-L1CAM, 1 μg/ml) diluted in blocking buffer, followed by the incubation with Cy3 donkey anti-Rabbit (1:600, Listarfish) secondary antibodies (45 min at RT). Finally, samples were washed in PBS and nuclei counterstained with DAPI solution (0.2 μg/ml, Sigma-Aldrich). Confocal microscopy was performed with a Leica SP2 confocal microscope equipped with a motorized stage and violet (405 nm laser diode), blue (488 nm Argon), yellow (561 nm laser diode), and red (633 nm HeNe laser) excitation laser lines. For quantification purposes, at least five fields for each condition were counted and the average of the positive/negative cells was calculated. The statistical difference among groups was determined as described in the Statistical analysis section.

Immunohistochemistry

Fresh tissue samples were obtained upon informed consent from patients undergoing surgery at the Gynecology Division of the European Institute of Oncology (Milan). Sample collection was performed under the protocol n. R789-IEO approved by the Ethics Committee of the European Institute of Oncology. The immunohistochemical analysis of L1CAM expression was carried out on a panel of high-grade serous ovarian carcinoma. Fresh samples were 4% PFA-fixed and paraffin embedded. After an overnight at 37°C, tumor sections (3 µm) were deparaffinized using Leica ST5020 Multistainer. Tissue sections were treated with the antigen unmasking solution EDTA pH 8 in pre-warmed water bath at 95°C for 50 min and then endogenous peroxidases were blocked using 3% Hydrogen peroxide solution (Carlo Erba). Tissue sections were incubated in blocking solution (TBS, 4% BSA, 0.05% Triton X-100) for 1 hr. Samples were then incubated for 2 hr with primary antibodies (anti-L1CAM, 1 μg/ml; anti-NOVA2 C-16, 1:100 SantaCruz; anti-CD31, 1:50 Abcam) followed by secondary antibodies incubation (Dako EnVision + System HRP Labelled Polymer or Goat-on-Rodent HRP Polymer from Biocare) for 30 min at RT. Dako chromogen substrate (Liquid DAB +Substrate Chromogen System Ref. K3468) or Vina Green Chromogen Kit (Biocare Medical) were used for signal detection. Samples were counterstained using Hematoxylin solution (Leica). Double immunohistochemistry for NOVA2 and L1CAM (using the same primary antibodies as above) was performed as previously described (Giampietro et al., 2015). Pictures of stained sections were acquired with the Aperio ScanScope XT instrument. For quantification of CD31 and NOVA2 staining, five different fields were counted for each section from five independent samples of normal ovaries or ovarian cancer. Vessel density was determined calculating the number of CD31-positive vessels per area unit (mm2) using a proprietary tool of the Aperio ImageScope software (Leica Biosystems Imaging).

RNA extraction, RT-PCR, RT-qPCR

Total RNA was isolated from both cultured cells and paraffin-embedded samples. RNA from cultured cells was obtained by using the RNeasy Mini Kit (QIAGEN), while tissue RNA was extracted with AllPrep DNA/RNA FFPE (QIAGEN) according to manufacturer’s instructions. cDNA was obtained starting from 500 to 1000 ng of total RNA with Superscript IV RT cDNA synthesis kit (Invitrogen) according to the manufacturer’s instructions and an aliquot (1–2 μl) of cDNA was then PCR-amplified (with GoTaq DNA Polymerase, Promega). The percentage of exon inclusion was calculated as the ratio between the intensity of the band of L1cam transcripts with the exon included and the total intensity of the L1cam bands. Splicing of L1CAM in human normal tissues (brain, heart, liver, kidney, lung and trachea, Clontech), normal human ovary and breast samples (Ambion) and human EC lines (HUVEC and hCMEC/D3) was analyzed with primers hL1E23_F and hL1E28_R (Supplementary file 1). RT-PCR analysis on FFPE samples of ovarian cancer tissue was performed with the primers hL1E24_F and hL1E26_R (Supplementary file 1) after selection of suitable areas of the tumors by a trained pathologist (GB). Splicing of L1cam in mouse tissues (brain, cortex, testis, lung, tongue and liver), freshly purified ECs from mouse lung and EC lines was analyzed with primers mL1E23_F and mL1E28_R (Supplementary file 1). Mouse tissues were obtained from Karolinska Institutet (Stockholm, Sweden) and IRCCS San Raffaele Scientific Institute (Milan, Italy), in accordance to Institutional Animal Care and Use Committees. Band intensity on agarose gel was quantified with the NIH Image J program (version 1.50i). All PCR products were verified by sequencing. For RT-qPCR experiments, cDNA samples were amplified with QuantiTect SYBR Green PCR (QIAGEN) by using LightCycler 480 (Roche). Target transcript levels were normalized to those of GAPDH or Ubb housekeeping genes. All primers used in RT-qPCR are listed in Supplementary file 1.

Crosslinking and immunoprecipitation (CLIP)

CLIP assay was performed as previously described (Paronetto et al., 2014; Paronetto et al., 2011). moEC were irradiated once with 150 mJ/cm2 in a Stratlinker 2400 at 254 nm. Cell suspension was centrifuged at 4000 rpm for 3 min, and pellet was incubated for 10 min on ice in lysis buffer [50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1% Igepal CA-630 (Sigma-Aldrich), 0.1% SDS, 0.5% sodium deoxycholate, 0.5 mM Na3VO4, 1 mM DTT, protease inhibitor cocktail (Sigma-Aldrich), and RNase inhibitor (Promega)]. Samples were briefly sonicated and incubated with 10 μl of 1:1000 RNase I (100 U/µL, Ambion) dilution and 2 μl of DNase (2 U/µL, Ambion) for 3 min at 37°C shaking at 1100 rpm, and then centrifuged at 15,000 g for 10 min at 4°C. One milligram of extract was immunoprecipitated using anti-NOVA2 antibody (C-16, Santa Cruz Biotechnology) or purified IgG (negative control) in the presence of protein A/G magnetic Dynabeads (Life Technologies). Immunoprecipitates were incubated overnight at 4°C under constant rotation. After stringent washes with high salt buffer (50 mM Tris-HCl, pH 7.4, 1 M NaCl, 1 mM EDTA, 1% Igepal CA-630, 0.1% SDS, 0.5% sodium deoxycholate), beads were equilibrated with PK buffer (100 mM Tris-HCl, pH 7.4, 50 mM NaCl, 10 mM EDTA). An aliquot (10%) was kept as input lysate, while the rest was treated with 50 µg Proteinase K and incubated for 20 min at 37°C shaking at 1100 rpm. 7 M urea was added to the PK buffer and incubated for further 20 min at 37°C shaking at 1100 rpm. The solution was collected and phenol/CHCl3 (Ambion) was added. After incubation for 5 min at 30°C shaking at 1100 rpm, phases were separated by centrifuging for 5 min at 13000 rpm at RT. The aqueous layer was transferred into a new tube and precipitated by addition of 0.5 μl glycoblue (Ambion), 3 M sodium acetate pH 5.5 and 100% ethanol. After mixing, the solution containing retained RNA was precipitated overnight at −20°C. RNA extracted from both the input material and the immunoprecipitates was then analyzed by RT-qPCR as described in the above paragraph. The binding was expressed as percentage of the input material.

Minigene splicing assay

HeLa cells were transiently co-transfected by using Lipofectamine 3000 with 500 ng of p-L1 WT minigene and either 500 ng of protein expression vectors (HA-NOVA2, T7-hnRNPA1 and T7-SRSF3) or the empty vector. Five-hundred ng of each mutated minigene (Mut3 and Mut5) were transfected with 250 ng of HA-NOVA2 expression vector plus 250 ng of the empty vector; as a control, we used 500 ng of the empty vector. Total RNAs were extracted from HeLa cells after 24 hr and analyzed by RT-PCR with primers p-L1_F and BGH_R annealing to mouse L1cam exon 25 and to the Bovine Growth Hormone (BGH) polyadenylation site, respectively. Primers are listed in Supplementary file 1.

L1-ΔTM recombinant protein production

For the production of secreted, recombinant L1-ΔTM (Figure 2—figure supplement 1), FreeStyle 293 F cells were cultured and transfected in FreeStyle medium. Four hours after transfection with pUPE.06.45-L1-ΔTM, Primatone RL (Sigma-Aldrich) was added to the culture medium at the final concentration of 0.6%. The culture medium was harvested 7 days after transfection by 10 min centrifugation at 1000 g. The supernatant containing secreted L1-ΔTM was loaded at 0.5 ml/min on a 1 ml StrepTrap HP column (GE Healthcare). The column was washed with 10 column volumes (cv) of buffer P, composed of 50 mM HEPES and 250 mM NaCl pH 7.5. Elution was performed with 1 cv of the same buffer supplemented with 5 mM d-Desthiobiotin (Sigma-Aldrich). After elution, protein was incubated overnight at 4°C with TEV protease to remove the N-terminal affinity tags. The resulting protein was concentrated by centrifugation using Vivaspin Turbo 15, 100000 MWCO centrifugal filters (Sartorius) to a volume less than 500 μl and then loaded onto a Superdex 200 10/300 GL (GE Healthcare) gel filtration column equilibrated with buffer P. The elution peak corresponding to L1-ΔTM as judged by SDS-PAGE analysis was collected and concentrated to 1 mg/ml. The purified sample was then flash-frozen in liquid nitrogen and stored at −80°C until further usage.

Conditioned medium production

Confluent mouse ECs were washed three times with PBS 1x and cultured for 48 hr in the following medium: DMEM High Glucose, 2 mM L-glutamine, 100 U/l penicillin streptomycin, 1 mM sodium pyruvate, 25 mM HEPES, 0.5% FBS. Conditioned media (CM) were collected and centrifuged for 10 min to remove cell debris.

In vitro tube formation assay

A Matrigel-based tubulogenesis assay was performed to assess the ability of ECs to form an organized capillary-like network. Before proceeding with the assay, 96-well plate was coated with 50 μl/well of Growth Factor-Reduced Matrigel (BD Biosciences) and left for 1 hr at 37°C for gelification. To assess the cell-autonomous effect of L1-ΔTM, transduced moEC were plated on Matrigel-coated plates in complete growth medium. Tube-like structures were manually counted under the microscope after 24 hr.

To assess the paracrine effect of L1-ΔTM, confluent moEC were cultured overnight in the same medium used for CM production. The day after, moEC were seeded on polymerized Matrigel-coated wells in the appropriate CM or, where indicated, were treated with different concentrations of recombinant, purified L1-ΔTM. After 8 hr of incubation at 37°C, tubes-like structures were counted under the microscope.

To block L1-ΔTM activity, transduced moEC were pre-incubated for 1 hr at 37 ˚C with 10 µg/ml of an anti-L1CAM blocking antibody [clone 324 (Di Sciullo et al., 1998; Appel et al., 1993)] or with control rat IgG (Sigma-Aldrich). Cells were then seeded on Matrigel-coated wells in complete growth medium containing 10 µg/ml of anti-L1CAM 324 (or control rat IgG) and tube-like structures were counted manually 8 hr later under the microscope. Where indicated, transduced moEC were pre-incubated for 1 hr at 37 ˚C with FGFR inhibitor PD173074 at a final concentration of 70 nM. Cells were then seeded on Matrigel-coated wells in normal growth medium containing 70 nM of PD173074 and tube-like structures were counted manually after 8 hr under the microscope. Both image acquisition and cell counts were performed using EVOS FL Imaging System. Cells were plated in triplicate (technical replicates) and the experiment was performed three times (biological replicates).

Matrigel plug

All animal studies were performed following a protocol approved by the fully authorized animal facility of European Institute of Oncology and by the Italian Ministry of Health (as required by the Italian Law) (IACUC n. 1256/2015) and in accordance to EU directive 2010/63. The sample size estimation was based on previous studies and pilot experiments. C57Bl/6 mice were injected into the right flank with 200 μl of CM derived from mouse ECs expressing either L1-ΔTM or L1-FL or from control ECs in a final volume of 600 μl of Growth Factor-Reduced Matrigel (BD Biosciences). Matrigel containing 0.5 μg of FGF2 was used as positive control. Groups were composed by three mice for each construct. Plugs were removed 7 days after injection, fixed in 4% PFA and paraffin embedded. Sections from fixed plugs were stained for CD31 as described above. The number of CD31-positive vessels that invaded Matrigel plugs were evaluated by manual counting of five different fields per section using Axioskop two microscope (Leica Biosystems).

Survival analysis

To determine the prognostic relevance of NOVA2, overall survival curves of ovarian cancer patients were built analyzing the GDC TCGA dataset with the UCSC Xena web tool (http://xena.ucsc.edu/). Survival plots were drawn using the Kaplan-Meier method and patients were stratified according to NOVA2 expression using median as threshold value. The log-rank Mantel-Cox test was employed to determine any statistical difference between the survival curves of the cohorts.

Analysis of RNA-seq data from NOVA2-knockdown ECs

We previously performed RNA sequencing on NOVA2-knockdown moEC versus their parental control (Giampietro et al., 2015). To capture additional NOVA2-regulated AS events in that experimental system, we first aligned each of the four samples independently (two control and two knockdown replicates) with the Vertebrate Alternative Splicing and Transcription Tools (vast-tools) (Tapial et al., 2017) (https://github.com/vastgroup/vast-tools). Then both replicates of each experimental point were pooled with vast-tools merge using the default parameters. This increased markedly the read coverage at each of the exon-exon junctions per experimental condition. Then, we applied vast-tools compare to perform a differential splicing analysis between the pooled control and the pooled knockdown samples, using a cutoff of delta percent spliced in (ΔPSI) of 15 with default settings.

Statistical analysis and data reporting

Independent experiments were considered as biological replicates. When performed, technical replicates deriving from the same biological replicate were averaged. For in vivo experiments, each mouse represented one biological replicate. For staining of human tissues, each patient represented one biological replicate. Data are expressed as mean ±SEM, calculated from at least three independent experiments. Student’s two-tailed t test or ANOVA multiple comparison test, followed by Tukey’s post hoc analysis, were used to compare two or three or more groups, respectively, and to determine statistical significance (GraphPad Prism 5). The correlation between the expression of L1-ΔTM and tumor vessel density was assessed using the Spearman rank correlation coefficient.

Differences were considered significant at p<0.05. Asterisks correspond to p-value calculated by two-tailed, unpaired, t-test (*p<0.05, **p<0.01, ***p<0.001). The sample size estimation was based on previous studies and pilot experiments.

References

  1. 1
  2. 2
  3. 3
  4. 4
  5. 5
  6. 6
  7. 7
  8. 8
  9. 9
  10. 10
  11. 11
  12. 12
    Developing global insight into RNA regulation
    1. RB Darnell
    (2006)
    Cold Spring Harbor Symposia on Quantitative Biology 71:321–327.
    https://doi.org/10.1101/sqb.2006.71.002
  13. 13
    Vascular Morphogenesis: Methods and Protocols
    1. L Di Blasio
    2. F Bussolino
    3. L Primo
    (2014)
    41–47, Three-dimensional in vitro assay of endothelial cell invasion and capillary tube morphogenesis, Vascular Morphogenesis: Methods and Protocols, Springer, 10.1007/978-1-4939-1462-3_4.
  14. 14
  15. 15
  16. 16
  17. 17
  18. 18
  19. 19
  20. 20
  21. 21
  22. 22
  23. 23
  24. 24
  25. 25
  26. 26
  27. 27
  28. 28
  29. 29
  30. 30
  31. 31
  32. 32
  33. 33
  34. 34
  35. 35
  36. 36
  37. 37
    Expression of the cell adhesion molecules N-CAM and L1 in B16 melanoma cells
    1. D Linnemann
    2. E Bock
    (1986)
    Medical Biology 64:345–349.
  38. 38
  39. 39
  40. 40
  41. 41
  42. 42
  43. 43
  44. 44
  45. 45
  46. 46
  47. 47
  48. 48
  49. 49
  50. 50
  51. 51
  52. 52
  53. 53
  54. 54
  55. 55
  56. 56
  57. 57
  58. 58
  59. 59
  60. 60
  61. 61
  62. 62
  63. 63
  64. 64
  65. 65
  66. 66
  67. 67
  68. 68
  69. 69
  70. 70
  71. 71
  72. 72
  73. 73
  74. 74
  75. 75
  76. 76
  77. 77
  78. 78
  79. 79
  80. 80

Decision letter

  1. Jonathan A Cooper
    Senior Editor; Fred Hutchinson Cancer Research Center, United States
  2. Douglas L Black
    Reviewing Editor; University of California, Los Angeles, United States
  3. Patrick A Murphy
    Reviewer; UConn Health, United States

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

[Editors’ note: a previous version of this study was rejected after peer review, but the authors submitted for reconsideration. The first decision letter after peer review is shown below.]

Thank you for submitting your work entitled "A Novel L1 Isoform with Angiogenic Activity Generated by NOVA2-mediated Alternative Splicing" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Patrick A Murphy (Reviewer #1).

Based on discussions between the reviewers and editors and the individual reviews below, we regret that we are rejecting your paper for publication in eLife.

All the reviewers and editors agreed that the basic results characterizing Nova2 regulation of L1 splicing and its possible role in tumor angiogenesis were quite interesting. However, there was general agreement that several key issues regarding angiogenic effects of the splice variant were not well supported. In particular, it is important to distinguish effects of the soluble splice variant from those of the extracellular domain that is cleaved from the typical isoform. Similarly, it is not clear whether the increased L1 and Nova2 expression observed in tumors was simply due to their increased vascularization and not causal as is proposed. To resolve this, the expression of the L1 variant and Nova2 need better quantification in normal and tumor endothelium. These issues are fundamental to the model that the L1 splice variant is driving tumor angiogenesis. The reviewers raise other valid points below.

Reviewer #1:

This is an interesting follow-up on previous work by this group, which had revealed an important function for the Nova2 splice factor in vascular development. Here, they reveal a connection between Nova2 and a target splice event in L1cam, resulting in a form of the protein with altered cellular localization and function. Furthermore, Nova2, which like L1cam is fairly endothelial specific in ovarian tumors, correlates with tumor progression. Overall, the work is well done and exciting, but I think there are a few points which could be addressed in greater detail.

How much of the angiogenic properties of L1cam are due to altered splicing is a critical question. Rather than comparing angiogenic properties of supernatants of cells with exon25 deficient version of L1cam (L1-ΔTM) to vector control, I would have liked to see a comparison between cells expressing exon25 exclusive and inclusive versions of L1cam (L1- ΔTM and L1-FL). This would help address how much of the angiogenic effects observed are due to the change in splicing, rather than L1cam expression alone. It would be best to do these experiments in the matrigel plug assay, which more closely indicates angiogenic function than the tubulogenesis assay (fibroblasts and cancer cells also form tubules on matrigel, PMID: 11806243).

A link between FGF and Exon25- negative L1cam is suggested but not confirmed by the experiments. Is there a change in FGF signaling that can be defined by immunofluorescence or Western blot? For example, could a difference in FGF receptor expression or phosphorylation be detected following treatment with soluble L1cam? One interpretation is that the FGF and L1cam signaling pathways are parallel inputs in the tube formation assay where both are required for tubulogenesis. Given these concerns and the use of the in vitro tube formation assay alone as an indication of their cross-talk, the claim that "L1-ΔTM promotes angiogenesis via FGFR1" seems overstated.

Authors allude to data on increased secretion of L1cam in endothelial cells (Discussion, first paragraph), but I do not see a data reference for this observation. Can they measure soluble L1cam with the C-terminus in the supernatant of their endothelial cell lines and some other L1 expressing cells which do not make the L1-ΔTM isoform? Or can they measure this in their endothelial cells with and without Nova2 suppression? This would help alleviate concerns about any artifacts due to overexpression of L1cam in moECs.

If L1-ΔTM is specific to tumor endothelium, it is not surprising that tumor levels are correlated with vascular density. It would be more meaningful if the endothelial levels of L1-ΔTM expression were correlated with tumor vascularization. There is some heterogeneity in the L1-ΔTM expression levels in isolated tumor endothelial cells (Figure 5C), do those with lower L1-ΔTM come from less vascular tumors? Do they have lower NOVA2 expression? Perhaps that is what the authors looked at already, but this was not clear to me.

Reviewer #2:

L1 (L1CAM gene) is a cell-surface glycoprotein that undergoes ectodomain shedding via proteolytic cleavage at a membrane proximal site. This soluble ectodomain has previously been implicated in a wide array of cell signaling and adhesion processes involved in tumorigenesis.

In this manuscript, Angiolini et al. identify a novel isoform of L1CAM with exon 25 skipped, termed L1-ΔTM. Because exon 25 contains the entire transmembrane domain of the protein, skipping of this exon results in a L1 protein isoform that no longer localizes on the cell surface and instead is found both in the cytoplasm and secreted into the extracellular space. The authors investigate whether this secreted full-length protein isoform of L1 (which does not undergo proteolytic cleavage and maintains the cytoplasmic tail) drives a pro-angiogenic program that supports aggressive tumorigenesis. They identify the well-characterized splicing factor, Nova2, as an important factor in promoting skipping of exon 25.

This manuscript presents a novel alternative (non-proteolytic) mechanism for the release of the L1 ectodomain into the extracellular space and characterizes the role of Nova2 in producing this isoform. The observation that this novel splice variant occurs in endothelial cells and its regulation by Nova2, as well as the exogenous expression of the full-length and L1-ΔTM isoforms to produce glycosylated transmembrane and soluble proteins, respectively, are all compelling pieces of data. However, the major flaw in the manuscript is the lack of physiological evidence that the angiogenic effects observed upon overexpression of L1-ΔTM are specific to the isoform itself, or whether a similar pro-angiogenic affect would also result from overexpressing full length L1. Indeed, overexpression of membrane bound L1 (FL-L1) resulted in release of L1 into the extracellular space (Figure 2D), presumably through proteolytic cleavage which would presumably have the same effect.

The difficulty in separating the effect of the cleaved full-length protein compared to the exon 25 skipped splice variant is acknowledged. The authors could address this issue by generating a mutation in the proteolytic cleavage site that would render it inactive and determine whether the splicing variant can overcome this deficiency.

A separate concern related to this criticism is that the manuscript relies almost entirely on a gain-of-function system. It is unclear how the level of overexpression of the L1-ΔTM isoform relates to endogenous expression either in cell lines or tumors, and without the L1-FL controls, one cannot assess the relative potency of this isoform being expressed.

Reviewer #3:

The manuscript by Angiolini et al. describes a novel alternatively spliced isoform of the cell-surface adhesion molecule L1CAM in endothelial cells (ECs). This novel isoform lacking exon 25, lacks the transmembrane domain, creating a soluble form of L1CAM. The authors show that this soluble L1-ΔTM isoform enhances the formation of tubes in mouse endothelial cells and this is inhibited by an inhibitor of FGFR1 signaling. They show direct binding of NOVA2 to exon 25 of the L1CAM transcript and in a splicing assay, show NOVA2 to be responsible for promoting skipping of L1CAM exon 25. Finally, they show a correlation between higher vessel density in L1-ΔTM-positive ovarian tumors, implying that alternative splicing of L1CAM correlates with tumor vascularization, supporting a proangiogenic function for L1-ΔTM.

In general this manuscript is well written and easy to follow. The experimental data is clearly presented. The novelty of this report is the identification of a cell adhesion molecule converted into a soluble angiogenic factor through alternative splicing. This represents not only a novel target for future antiangiogenic strategies but also can be used for diagnostic and prognostic purposes. However, there are some things lacking in the paper that need to be addressed.

1) Even though NOVA2 is an important splicing factor in EC cells, there is a bit of a logic "jump" caused by focusing only on NOVA2 as the regulator of L1CAM. The authors need to show other possible splicing factors from the SFmap/SpliceAid analysis, and possibly knockdown several of them (e.g. SRSF1, SRSF2, PTBP1, in addition to hnRNP A1) in their minigene splicing assay or RT-PCR on the endogenous transcripts.

2) Figure 5 The authors need to normalize the amount of L1CAM and NOVA2 to the amount of blood vessels in the IHC. If any blood vessel expresses L1CAM and NOVA2 then it is only a measure of higher vascularity in the tumor. The relevant question is if the blood vessels in the tumor express higher L1CAM and NOVA2 than normal? Also, it seems that additional cells are stained with NOVA2 (epithelial cells or other cells?) which do not express L1CAM.

3) In order to claim that the blood vessels in the tumor express more NOVA2 and L1CAM isoform, the authors need to show that in the presence of the cancer cells the EC cells express higher levels of NOVA2. Maybe this is through secretion of factors from the cancer cells or direct interaction, but the claim is not proven until some causative experiment is performed.

4) Figure 4—figure supplement 3B and C should show endogenous levels of Nova2 in the transfected cells. The authors write in Materials and methods "Since NOVA2 expression is regulated by EC density (Giampietro et al., 2015), for the analysis of L1 splicing, NOVA2-knockdown moEC were used as confluent monolayers (500,000 cells in 35-mm Petri dishes), whereas moEC overexpression HA-tagged NOVA2 were tested at low density (500,000 cells in 100-mm Petri dishes)." If the expression is sensitive to cell density it should be shown for the specific experiments.

5) Figure 4—figure supplement 3F. The effect of Nova2 overexpression on L1CAM splicing in human cells (82% inclusion) is not as strong as in mouse cells Figure 4B (27.2% inclusion). Can the authors provide an explanation for that?

[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]

Thank you for submitting your article "A Novel L1 Isoform with Angiogenic Activity Generated by NOVA2-mediated Alternative Splicing" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by Douglas Black as Reviewing Editor and Jonathan Cooper as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Patrick A Murphy (Reviewer #1).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

This manuscript is a resubmission of a previously rejected study. The reviewers agreed that the previous version was of significant interest but all had major criticisms and experiments that they felt were needed to support its conclusions. All of the reviewers now agree that the authors have made major changes that strengthen the paper. Assuming that the authors can adequately address the remaining points in these reviews, the editors are pleased to accept this paper for publication in eLife.

Reviewer #1:

The addition of new data has solidified key conclusions and addressed my original concerns. With these new pieces of data, the authors make a compelling argument that a reduction in the inclusion of exon 25, which is mediated by Nova2 in the endothelium, promotes the expression of a secreted L1 variant with angiogenic properties beyond the constitutive form of L1. I particularly like the addition of Supplementary Figure 2M and N, showing that a morpholino suppressing L1 exon 25 inclusion results in increased secretion of soluble L1. I think this should be considered for the main figures.

Reviewer #2:

This manuscript is a resubmission of a previously reviewed work that all reviewers agreed presents an important and novel biological mechanism in which alternative splicing essentially replaces proteolytic cleavage to release a polypeptide that is otherwise integrally associated with the plasma membrane. The authors have addressed most of the initial reviewers' comments and the paper is clearly much stronger now from the addition of new data and textual clarifications.

I had originally suggested a making a mutation in the proteolytic site – Figure 2A showing the putative ADAM cleavage site as a box implies some knowledge of where cleavage occurs (hence the suggested experiment, as there are only 6 amino acids in the extracellular domain of that region). If it is not clear where the cleavage occurs (and which enzymes are responsible), this figure is a bit misleading. The difficulties the authors cite regarding performing the suggested experiments are reasonable, but perhaps the figure could be clarified.

The immunofluorescence data added to Figure 2C is much improved with regard to clearly showing the subcellular localization of each isoform. The data that has been added to Figure 3 (and in particular Figure 3F) are instrumental in addressing both my concerns and those of reviewer #1, this experiment adds a great deal of support to the central hypothesis of this paper.

The authors have taken the criticism that the previous manuscript was overly dependent on gain-of-function experiments constructively in adding the new data presented in Figure 2—figure supplement 1. These experiments provide an elegant alternative approach to overexpression of individual isoforms by perturbing the splicing of the endogenous gene. This is a well-conceived and executed experiment.

Figure 5B solidifies the assertion that NOVA2 and L1 are colocalized. This is a strong piece of evidence for the physiological relevance of the proposed mechanism within tumors.

In summary, with this resubmission the authors have more than satisfactorily addressed the major points of concern raised in the original manuscript.

Reviewer #3:

The manuscript "A novel L1 isoform with angiogenic activity generated by Nova-2 mediated alternative splicing" has been revised. The authors have supplied additional data in several figures, new supplementary figures and table and two figures in their response to the reviewers. In addition, they have addressed the concerns of the reviewers in their letter.

They have analyzed a cohort of 13 ovarian cancer samples for vessel density and expression levels of L1-ΔTM. They show that the levels of mRNA correlate with the degree of tumor vascularization (Figure 5D). They have also redone the immunohistochemistry of NOVA2 in EC cells (Figure 5B) showing that the immunoreactivity of NOVA2 is restricted to the nuclei of vascular ECs.

The authors did not completely address point 3. Thus, the connection between L1-ΔTM and vascularization is left at the level of correlation rather than a causative effect. The authors should at least say that further experiments are required to determine if this a direct effect of L1-ΔTM.

https://doi.org/10.7554/eLife.44305.025

Author response

[Editors’ note: the author responses to the first round of peer review follow.]

Reviewer #1:

This is an interesting follow-up on previous work by this group, which had revealed an important function for the Nova2 splice factor in vascular development. Here, they reveal a connection between Nova2 and a target splice event in L1cam, resulting in a form of the protein with altered cellular localization and function. Furthermore, Nova2, which like L1cam is fairly endothelial specific in ovarian tumors, correlates with tumor progression. Overall, the work is well done and exciting, but I think there are a few points which could be addressed in greater detail.

How much of the angiogenic properties of L1cam are due to altered splicing is a critical question. Rather than comparing angiogenic properties of supernatants of cells with exon25 deficient version of L1cam (L1-ΔTM) to vector control, I would have liked to see a comparison between cells expressing exon25 exclusive and inclusive versions of L1cam (L1-ΔTM and L1-FL). This would help address how much of the angiogenic effects observed are due to the change in splicing, rather than L1cam expression alone. It would be best to do these experiments in the matrigel plug assay, which more closely indicates angiogenic function than the tubulogenesis assay (fibroblasts and cancer cells also form tubules on matrigel, PMID: 11806243).

We have performed the matrigel plug assay as recommended by the reviewer. As shown in Figure 3F, and described in the last paragraph of the subsection “L1-ΔTM regulates endothelial cell function”, the conditioned medium (CM) from L1-ΔTM- expressing ECs was significantly more angiogenic than the CM from L1-FL-expressing cells. Incidentally, we obtained similar results also in the tubulogenesis assay (see Author response image 1). Based on the reviewer’s concern about the tube formation assay, we did not include these in vitrodata in the revised manuscript, and just mentioned them as data not shown in the second paragraph of the aforementioned subsection.

These additional experiments indicated that L1-ΔTM per seis endowed with angiogenic properties, while the mere expression of L1cam has little effect, further supporting the biological relevance of the splicing of exon 25 in endothelial cells.

Author response image 1
L1-ΔTM promotes endothelial tube formation.

Parental moEC were subjected to tube formation assays in the presence of the CM from moEC transduced with either the empty vector, L1-FL or L1- ΔTM. The CM from L1- ΔTM-expressing cells exhibited higher in vitroangiogenic potential than that from L1-FL- expressing cells or from control cells. **p<0.01; *p<0.05.

A link between FGF and Exon25- negative L1cam is suggested but not confirmed by the experiments. Is there a change in FGF signaling that can be defined by immunofluorescence or Western blot? For example, could a difference in FGF receptor expression or phosphorylation be detected following treatment with soluble L1cam? One interpretation is that the FGF and L1cam signaling pathways are parallel inputs in the tube formation assay where both are required for tubulogenesis. Given these concerns and the use of the in vitro tube formation assay alone as an indication of their cross-talk, the claim that "L1-ΔTM promotes angiogenesis via FGFR1" seems overstated.

As suggested by the reviewer, we investigated whether soluble L1-ΔTM affects FGFR phosphorylation. Treating parental moEC with recombinant L1-ΔTM resulted in increased phospho-FGFR1, consistent with the L1-ΔTM-induced activation of FGFR1. These observations have been included in the Results (Figure 3G and subsection “FGFR1 signaling is required for L1-ΔTM-induced tube formation”).

The reviewer is correct in pointing out that FGFR1 and L1CAM might activate parallel pathways that are both involved in tube formation. However, the observation that the FGFR inhibitor PD173074 reduces the tubulogenesis of L1-ΔTM-expressing moEC to the level of control cells (but has no effect on the L1-ΔTM-independent tube formation in control moEC), strongly suggests that in the context of tubulogenesis FGFR1 signaling acts as an effector of L1-ΔTM. Nevertheless, we agree with the reviewer’s concern about the overstatement and have modified the sentence accordingly (“FGFR1 signaling is required for L1-ΔTM- induced tube formation”).

Authors allude to data on increased secretion of L1cam in endothelial cells (Discussion, first paragraph), but I do not see a data reference for this observation. Can they measure soluble L1cam with the C-terminus in the supernatant of their endothelial cell lines and some other L1 expressing cells which do not make the L1-ΔTM isoform? Or can they measure this in their endothelial cells with and without Nova2 suppression? This would help alleviate concerns about any artifacts due to overexpression of L1cam in moECs.

With regard to the concern about artifacts, we would argue that the data shown in Figure 2—figure supplement 1 reduce significantly the risk of artifacts due to the overexpression of L1CAM. The figure, indeed, shows in both moEC and luEC that the ectopic expression of L1-FL per se (shown in Figure 2—figure supplement 1A, lower panel, and Figure 2—figure supplement 1B, right upper panel) does not result in the secretion of significant amounts of L1 with the C-terminus. The latter is detected occurs only in the CM of L1-ΔTM- expressing cells (Figure 2—figure supplement 1A, upper panel, and Figure 2—figure supplement 1B, right lower panel), supporting the specificity of our results.

Nevertheless, the reviewer’s suggestion to compare endothelial cells with another L1- expressing cell type was very insightful. We compared mouse lung-derived EC, which express endogenous L1-ΔTM (Figure 1C, “lu2EC”) and the mouse melanoma cell line B16, known to express L1 (Linnemann and Bock, 1986; Magrini et al., 2014). We first verified by RT-PCR that B16 cells do not express the L1-ΔTM isoform (Figure 2—figure supplement 1E), which is consistent with the lack of Nova2 expression (Figure 2—figure supplement 1E), and then assayed for the secretion of C- terminus-containing L1 in the conditioned medium. Despite B16 cells express much higher levels of endogenous, cell-associated L1 than lu2EC (Figure 2—figure supplement 1F), no protein was detectable in their conditioned medium with the antibody against the cytoplasmic tail, while lu2EC secreted high amounts of C-terminus-containing L1 (Figure 2—figure supplement 1F). Together with the other data presented in the manuscript, these findings (described in the fourth paragraph of the subsection “Alternative splicing generates a novel soluble form of L1”) strongly support the increased secretion of L1- ΔTM in endothelial cells.

If L1-ΔTM is specific to tumor endothelium, it is not surprising that tumor levels are correlated with vascular density. It would be more meaningful if the endothelial levels of L1-ΔTM expression were correlated with tumor vascularization. There is some heterogeneity in the L1-ΔTM expression levels in isolated tumor endothelial cells (Figure 5C), do those with lower L1-ΔTM come from less vascular tumors? Do they have lower NOVA2 expression? Perhaps that is what the authors looked at already, but this was not clear to me.

We agree with the reviewer that “it would be more meaningful if the endothelial levels of L1-ΔTM expression were correlated with tumor vascularization”. Unfortunately, we could not assess the vascularization of the same tumors used to derive endothelial cells, as proposed by the reviewer, since the entire surgical specimens were processed to obtain the ECs themselves. Nevertheless, since our data strongly suggest that tissue expression of L1-ΔTM is largely confined to vascular endothelium (e.g., Figure 1B, C and Figure 5B), we performed RT-PCR for L1-ΔTM on a cohort of 17 archival samples of ovarian cancer. The same specimens were also stained for CD31 to measure vessel density. As shown in Figure 5D, the levels of L1-ΔTM mRNA correlated with the degree of tumor vascularization (r=0.84, p<0.001).

Reviewer #2:

[…] This manuscript presents a novel alternative (non-proteolytic) mechanism for the release of the L1 ectodomain into the extracellular space and characterizes the role of Nova2 in producing this isoform. The observation that this novel splice variant occurs in endothelial cells and its regulation by Nova2, as well as the exogenous expression of the full-length and L1-ΔTM isoforms to produce glycosylated transmembrane and soluble proteins, respectively, are all compelling pieces of data. However, the major flaw in the manuscript is the lack of physiological evidence that the angiogenic effects observed upon overexpression of L1-ΔTM are specific to the isoform itself, or whether a similar pro-angiogenic affect would also result from overexpressing full length L1. Indeed, overexpression of membrane bound L1 (FL-L1) resulted in release of L1 into the extracellular space (Figure 2D), presumably through proteolytic cleavage which would presumably have the same effect.

We thank reviewer #2 for her/his positive comments and for the constructive criticisms that allowed us to improve our manuscript.

With regard to the L1-ΔTM-specific effect, as specified also in our reply to reviewer #1, we have performed both in vivo matrigel plug and in vitro tube formation assays to compare cells expressing L1-ΔTM vs. full-length L1. As shown in Figure 3F and described in the last paragraph of the subsection “L1-ΔTM regulates endothelial cell function”, the conditioned medium (CM) from L1-ΔTM-expressing ECs was significantly more angiogenic than the CM from L1-FL-expressing cells. Similar results were obtained in the tubulogenesis assay (see Author response image 1). As recommended by reviewer #1, only the results of the Matrigel plug assay have been included in the manuscript.

The difficulty in separating the effect of the cleaved full-length protein compared to the exon 25 skipped splice variant is acknowledged. The authors could address this issue by generating a mutation in the proteolytic cleavage site that would render it inactive and determine whether the splicing variant can overcome this deficiency.

We agree with the reviewer that this would be a very elegant and appropriate strategy. However, it would be quite a challenging task, given that the proteolytic cleavage of L1 can be carried out by a plethora of proteases (ADAM10, ADAM17, plasmin, trypsin, proprotein convertase PC5A, etc.). Moreover, L1 shedding has been investigated mostly in neurons, and the enzymes involved in the cleavage of L1 in endothelial cells remain elusive. Therefore, it would be quite difficult and time-consuming to identify such enzymes and perform the mutagenesis suggested by reviewer #2, and we feel that such an effort would fall out of the scope of our manuscript.

A separate concern related to this criticism is that the manuscript relies almost entirely on a gain-of-function system. It is unclear how the level of overexpression of the L1-ΔTM isoform relates to endogenous expression either in cell lines or tumors, and without the L1-FL controls, one cannot assess the relative potency of this isoform being expressed.

We thank the reviewer for asking these important controls, which lent further support to our conclusions. As described above in our response to reviewer #1, we have included the requested L1-FL controls. The results showed that L1-ΔTM-containing conditioned medium (CM) has a markedly higher angiogenic potential than L1-FL CM (Figure 3F and Author response image 1).

To address the concern on the manuscript relying almost entirely on a gain-of-function system, we have added new data on an endogenous system. The lung-derived endothelial cells lu2EC, which express both NOVA2 and L1-ΔTM endogenously (Figures 1C and Figure 2—figure supplement 1E), were treated with a morpholino oligonucleotide that selectively interfered with the inclusion of exon 25 (Figure 3E). As shown in Figure 3F (left), this resulted in increased skipping of exon 25 and release of endogenous L1-ΔTM in the extracellular compartment. Importantly, ECs exposed to the CM from morpholino-treated cells exhibited higher tube-forming activity than those exposed to control CM (Figure 3F, right). These observations validated in an endogenous system our observations on the angiogenic activity of AS-generated L1-ΔTM.

Reviewer #3:

[…] 1) Even though NOVA2 is an important splicing factor in EC cells, there is a bit of a logic "jump" caused by focusing only on NOVA2 as the regulator of L1CAM. The authors need to show other possible splicing factors from the SFmap/SpliceAid analysis, and possibly knockdown several of them (e.g. SRSF1, SRSF2, PTBP1, in addition to hnRNP A1) in their minigene splicing assay or RT-PCR on the endogenous transcripts.

As outlined in more details in the Results (paragraph “NOVA2 controls alternative splicing of L1-ΔTM in ECs”), the SFmap analysis identified NOVA2, hnRNP A1 and SRSF3 as possible candidate regulators of L1cam exon 25 splicing (Figure 4—figure supplement 1A). By using the minigene- based splicing assay as suggested by the reviewer, we found that the skipping of L1cam exon 25 occurred only upon overexpression of NOVA2, but not with hnRNP A1 or SRSF3 (Figure 4B and Figure 4—figure supplement 1B).

To further probe the specificity of NOVA2, we also tested SRSF1, as an additional AS regulator, and two known splicing repressors, hnRNP M and hnRNP A2/B1. None of these additional candidates was able to affect skipping of L1cam exon 25 (data not included in the revised manuscript but shown in Author response image 2).

The rationale for investigating NOVA2-mediated AS of L1cam was also supported by the analysis of the RNA-seq data from NOVA2-knockdown ECs (Giampietro et al., 2015), which revealed L1cam exon 25 as a novel target of NOVA2 in ECs (subsection “NOVA2 controls alternative splicing of L1-ΔTM in ECs”, second paragraph, and Supplementary file 2).

Author response image 2
Evaluation of candidate splicing regulatory factors (SRFs) on L1cam splicing.

AS of the p-L1 wild- type WT minigene co-transfected in HeLa cells with either HA-NOVA2, FLAG-hnRNP A2B1 (FLAG-A2B1), GFP- hnRNP M (GFP-M) or the empty vector. Top panels show that, in contrast to NOVA2, none of the other SRFs affected the splicing of L1cam exon 25. Bottom panels show the ectopic expression of each SRF as revealed by western blotting with the indicated antibodies (α-Tubulin as loading control).

2) Figure 5 The authors need to normalize the amount of L1CAM and NOVA2 to the amount of blood vessels in the IHC. If any blood vessel expresses L1CAM and NOVA2 then it is only a measure of higher vascularity in the tumor. The relevant question is if the blood vessels in the tumor express higher L1CAM and NOVA2 than normal? Also, it seems that additional cells are stained with NOVA2 (epithelial cells or other cells?) which do not express L1CAM.

Figure 5A (left panel) shows indeed the percentage of NOVA2-positive vessels over the total number of vessels (determined by CD31 staining). We have now clarified this in the legend to Figure 5A as well as on the Y-axis of the graph. In addition, we now provide analogous measurements also for L1-positive vessels, confirming that they are markedly increased in cancer samples (Figure 5A, right panel).

Reviewer #3 argues that “if any blood vessel expresses L1CAM and NOVA2 then it is only a measure of higher vascularity in the tumor”. This concern was somehow similar to the last major comment of reviewer #1. To investigate in more detail the possible correlation between the expression of L1-ΔTM and tumor angiogenesis, we have analysed a cohort of 13 ovarian cancer samples both for vessel density and for the expression levels of L1-ΔTM (for more details, see our reply to the last major comment of reviewer #1). As shown in Figure 5D, the levels of L1-ΔTM mRNA correlated with the degree of tumor vascularization (r=0.84, p<0.001).

As for the expression of NOVA2 in additional cell types, we acknowledge that from the original IHC images one could hypothesize the presence of NOVA2 in non-endothelial cells. We have investigated that issue in more detail and found out that the faint cytosolic staining was due to non-specific interactions of the antibodies. Indeed, we have now optimized the immunohistochemistry conditions (using Viva Green instead of diaminobenzidine as the chromogenic substrate) and are able to show that the immunoreactivity of NOVA2 is restricted to the nuclei of vascular ECs (Figure 5B and Figure 5—figure supplement 2A). Finally, the vessel-specific immunoreactivity of NOVA2 is also shown in the images taken from the Human Protein Atlas website (Figure 5—figure supplement 1B).

3) In order to claim that the blood vessels in the tumor express more NOVA2 and L1CAM isoform, the authors need to show that in the presence of the cancer cells the EC cells express higher levels of NOVA2. Maybe this is through secretion of factors from the cancer cells or direct interaction, but the claim is not proven until some causative experiment is performed.

We are afraid we did not understand completely the reviewer’s comment. In particular, we do not see the causal link between the effect of cancer cells on the endothelial expression of NOVA2 and our “claim that the blood vessels in the tumor express more NOVA2 and L1CAM isoform”. Along the same line, it is unclear to us why “the claim is not proven until some causative experiment [on the role of cancer cells] is performed”. The increased expression of NOVA2 and L1CAM in ovarian cancer vasculature is illustrated in Figure 5A, where we show that tumors contain 6 and 4-fold more vessels positive for NOVA2 and L1CAM, respectively, than their normal tissue counterpart. This phenomenon is independent on whether and how cancer cells regulate the expression of the two proteins in the endothelium.

Having said that, we agree with the reviewer about the relevance of understanding the mechanisms that regulate the expression of NOVA2 in tumor-associated ECs. Nevertheless, we feel that investigating this issue would fall out of the scope of the present study. The experiments suggested by the reviewer, in addition, would be quite time-consuming in that they should not be limited to the cancer cells themselves, but should rather be extended to other components of the tumor microenvironment. It is conceivable, indeed, that different cell types and/or other microenvironmental factors account for (or at least contribute to) the induction of NOVA2 in the cancer vasculature. Along this line, a very recent paper reported that NOVA2 is upregulated in colorectal carcinoma vasculature and that endothelial NOVA2 expression is induced upon exposure to hypoxia (Gallo et al., 2018), a condition shared by many solid tumors. This paper is now cited in our Discussion (sixth paragraph).

4) Figure 4—figure supplement 3B and C should show endogenous levels of Nova2 in the transfected cells. The authors write in Materials and methods "Since NOVA2 expression is regulated by EC density (Giampietro et al., 2015), for the analysis of L1 splicing, NOVA2-knockdown moEC were used as confluent monolayers (500,000 cells in 35-mm Petri dishes), whereas moEC overexpression HA-tagged NOVA2 were tested at low density (500,000 cells in 100-mm Petri dishes)." If the expression is sensitive to cell density it should be shown for the specific experiments.

Figure 4—figure supplement 3A now shows the endogenous levels of Nova2 and L1-ΔTM in moEC cultured as sparse cells or at confluency. Confluent cells exhibit higher Nova2 expression as compared to sparse cells, at both mRNA and protein levels, thus demonstrating that Nova2 expression in moEC is sensitive to cell density, in agreement with previous data (Giampietro et al., 2015). The higher level of Nova2 in confluent moEC is mirrored by an increase in the exclusion of L1cam exon 25 (Figure 4—figure supplement 3A).

5) Figure 4—figure supplement 3F. The effect of Nova2 overexpression on L1CAM splicing in human cells (82% inclusion) is not as strong as in mouse cells Figure 4B (27.2% inclusion). Can the authors provide an explanation for that?

While it is possible that the discrepancy involves, at least in part, some species-specific features, also the origin of ECs may play a role in the relative efficiency of NOVA2 in L1CAM splicing. In this specific case, moEC derive from mouse lung, while hCMEC/D3 derive from human brain vessels. The heterogeneity of ECs from different districts, in fact, has been extensively documented and may reflect profoundly different phenotypic and functional properties (Potente and Mäkinen, Nat Rev Mol Cell Biol, 2017).

To further address the reviewer’s comment, we have reduced by shRNA the expression of endogenous NOVA2 in a different EC model, consisting of human umbilical vein ECs. As shown in Figure 4—figure supplement 3G, NOVA2 knockdown resulted in a marked decrease in L1CAM splicing (with the inclusion of exon 25 going from 72% to 100%). This supports the hypothesis that the splicing of exon 25 in response to the genetic manipulation of NOVA2 is quantitatively different indifferent EC types. Of note, these data suggest that the activity of NOVA2 is cell context- dependent even within the same cell type (i.e., endothelial cells).

[Editors' note: the author responses to the re-review follow.]

Reviewer #1:

The addition of new data has solidified key conclusions and addressed my original concerns. With these new pieces of data, the authors make a compelling argument that a reduction in the inclusion of exon 25, which is mediated by Nova2 in the endothelium, promotes the expression of a secreted L1 variant with angiogenic properties beyond the constitutive form of L1. I particularly like the addition of Supplementary Figure 2M and N, showing that a morpholino suppressing L1 exon 25 inclusion results in increased secretion of soluble L1. I think this should be considered for the main figures.

We thank reviewer #1 for his appreciation of the work we have done to improve our manuscript. We also thank him for his comments on the data presented in Supplementary Figure 2M and N. Following his suggestion, we have modified Figure 3 to include the morpholino data (Figure 3E and F). In order to avoid overcrowding of the figure, the microscope pictures of the tubes previously shown in Figure 3A have been moved to Figure 3—figure supplement 1A.

Reviewer #2:

[…] I had originally suggested a making a mutation in the proteolytic site – Figure 2A showing the putative ADAM cleavage site as a box implies some knowledge of where cleavage occurs (hence the suggested experiment, as there are only 6 amino acids in the extracellular domain of that region). If it is not clear where the cleavage occurs (and which enzymes are responsible), this figure is a bit misleading. The difficulties the authors cite in the letter regarding performing the suggested experiments are reasonable, but perhaps the figure could be clarified.

We agree with the reviewer that Figure 2A was misleading. Therefore, following the reviewer’s suggestion to clarify the figure and considering that our manuscript does not deal with the proteolytic cleavage of L1CAM, we have removed the box indicating the putative ADAM cleavage site.

Reviewer #3:

[…] The authors did not completely address point 3. Thus, the connection between L1-ΔTM and vascularization is left at the level of correlation rather than a causative effect. The authors should at least say that further experiments are required to determine if this a direct effect of L1-ΔTM.

We appreciate the reviewer’s thorough analysis of the new data that have been added in the revised manuscript. With regard to the last point, we have modified the Discussion to point out that further work should test the causal role of vascular L1-ΔTM in tumor vascularization (Discussion, sixth paragraph).

https://doi.org/10.7554/eLife.44305.026

Article and author information

Author details

  1. Francesca Angiolini

    Unit of Gynecological Oncology Research, Program of Gynecological Oncology, IEO, European Institute of Oncology IRCCS, Milan, Italy
    Present address
    GSK Vaccines Srl, Siena, Italy
    Contribution
    Conceptualization, Data curation, Writing—original draft, Writing—review and editing
    Contributed equally with
    Elisa Belloni and Marco Giordano
  2. Elisa Belloni

    Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche, Pavia, Italy
    Contribution
    Conceptualization, Data curation, Writing—original draft, Writing—review and editing
    Contributed equally with
    Francesca Angiolini and Marco Giordano
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6580-5553
  3. Marco Giordano

    Unit of Gynecological Oncology Research, Program of Gynecological Oncology, IEO, European Institute of Oncology IRCCS, Milan, Italy
    Contribution
    Conceptualization, Data curation, Writing—original draft, Writing—review and editing
    Contributed equally with
    Francesca Angiolini and Elisa Belloni
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6335-0030
  4. Matteo Campioni

    The Armenise-Harvard Laboratory of Structural Biology, Department of Biology and Biotechnology, University of Pavia, Pavia, Italy
    Contribution
    Data curation
    Competing interests
    No competing interests declared
  5. Federico Forneris

    The Armenise-Harvard Laboratory of Structural Biology, Department of Biology and Biotechnology, University of Pavia, Pavia, Italy
    Contribution
    Data curation
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7818-1804
  6. Maria Paola Paronetto

    Department of Movement, Human and Health Sciences, Università degli Studi di Roma "Foro Italico", Rome, Italy
    Contribution
    Data curation
    Competing interests
    No competing interests declared
  7. Michela Lupia

    Unit of Gynecological Oncology Research, Program of Gynecological Oncology, IEO, European Institute of Oncology IRCCS, Milan, Italy
    Contribution
    Data curation
    Competing interests
    No competing interests declared
  8. Chiara Brandas

    Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche, Pavia, Italy
    Contribution
    Data curation
    Competing interests
    No competing interests declared
  9. Davide Pradella

    1. Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche, Pavia, Italy
    2. Università degli Studi di Pavia, Pavia, Italy
    Contribution
    Data curation
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8649-7817
  10. Anna Di Matteo

    Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche, Pavia, Italy
    Contribution
    Data curation
    Competing interests
    No competing interests declared
  11. Costanza Giampietro

    FIRC Institute of Molecular Oncology, Milan, Italy
    Present address
    Laboratory of Thermodynamics in Emerging Technologies, Department of Mechanical and Process Engineering, ETH Zurich, Zurich, Switzerland
    Contribution
    Data curation
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5229-3835
  12. Giovanna Jodice

    Molecular Medicine Program, IEO, European Institute of Oncology IRCCS, Milan, Italy
    Contribution
    Data curation
    Competing interests
    No competing interests declared
  13. Chiara Luise

    Molecular Medicine Program, IEO, European Institute of Oncology IRCCS, Milan, Italy
    Contribution
    Data curation
    Competing interests
    No competing interests declared
  14. Giovanni Bertalot

    Molecular Medicine Program, IEO, European Institute of Oncology IRCCS, Milan, Italy
    Contribution
    Data curation
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4862-7705
  15. Stefano Freddi

    Molecular Medicine Program, IEO, European Institute of Oncology IRCCS, Milan, Italy
    Contribution
    Data curation
    Competing interests
    No competing interests declared
  16. Matteo Malinverno

    FIRC Institute of Molecular Oncology, Milan, Italy
    Contribution
    Data curation
    Competing interests
    No competing interests declared
  17. Manuel Irimia

    1. Centre for Genomic Regulation, The Barcelona Institute of Science and Technology, Barcelona, Spain
    2. Universitat Pompeu Fabra, Barcelona, Spain
    3. Institució Catalana de Recerca i Estudis Avançats, Barcelona, Spain
    Contribution
    Data curation, Funding acquisition
    Competing interests
    No competing interests declared
  18. Jon D Moulton

    Gene Tools LLC, Philomath, United States
    Contribution
    Resources
    Competing interests
    Affiliated with Gene Tools, LLC. Funding bodies had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4977-0802
  19. James Summerton

    Gene Tools LLC, Philomath, United States
    Contribution
    Resources
    Competing interests
    Affiliated with Gene Tools, LLC. Funding bodies had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.
  20. Antonella Chiapparino

    The Armenise-Harvard Laboratory of Structural Biology, Department of Biology and Biotechnology, University of Pavia, Pavia, Italy
    Contribution
    Data curation
  21. Carmen Ghilardi

    Laboratory of Biology and Treatment of Metastasis, IRCCS-Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy
    Contribution
    Data curation, Writing—review and editing
    Competing interests
    No competing interests declared
  22. Raffaella Giavazzi

    Laboratory of Biology and Treatment of Metastasis, IRCCS-Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy
    Contribution
    Data curation, Funding acquisition, Writing—review and editing
    Competing interests
    No competing interests declared
  23. Daniel Nyqvist

    Division of Vascular Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden
    Contribution
    Data curation
    Competing interests
    No competing interests declared
  24. Davide Gabellini

    Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute, Milan, Italy
    Contribution
    Data curation
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3811-4121
  25. Elisabetta Dejana

    1. FIRC Institute of Molecular Oncology, Milan, Italy
    2. Rudbeck Laboratory and Science for Life Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden
    Contribution
    Data curation, Writing—review and editing
    Competing interests
    Reviewing editor, eLife
  26. Ugo Cavallaro

    Unit of Gynecological Oncology Research, Program of Gynecological Oncology, IEO, European Institute of Oncology IRCCS, Milan, Italy
    Contribution
    Conceptualization, Data curation, Supervision, Funding acquisition, Writing—original draft, Project administration, Writing—review and editing
    Contributed equally with
    Claudia Ghigna
    For correspondence
    ugo.cavallaro@ieo.it
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0884-6460
  27. Claudia Ghigna

    Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche, Pavia, Italy
    Contribution
    Conceptualization, Funding acquisition, Writing—original draft, Project administration, Writing—review and editing
    Contributed equally with
    Ugo Cavallaro
    For correspondence
    arneri@igm.cnr.it
    Competing interests
    Consultant for Gene-Tools, LLC. Funding bodies had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0362-783X

Funding

Associazione Italiana per la Ricerca sul Cancro (IG-17395)

  • Claudia Ghigna

Worldwide Cancer Research (AICR 10-0091)

  • Ugo Cavallaro

Horizon 2020 Framework Programme (ERC-StG-LS2-637591)

  • Manuel Irimia

Ministerio de Economía y Competitividad (BFU2017-89201-P)

  • Manuel Irimia

Giovanni Armenise-Harvard Foundation

  • Federico Forneris

Ministero dell'Istruzione dell'Università e della Ricerca

  • Federico Forneris

Associazione Italiana per la Ricerca sul Cancro (IG-14622)

  • Ugo Cavallaro

Associazione Italiana per la Ricerca sul Cancro (IG-18853)

  • Raffaella Giavazzi

Associazione Italiana per la Ricerca sul Cancro (IG-18683)

  • Elisabetta Dejana

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

This paper is dedicated to the memory of our friend and colleague Prof. Giovanni Morrone. We are grateful to F Bianchi for gene expression analysis, S Pirroni for assistance with RNA extraction, A Canciani for assistance with L1-ΔTM cloning, V Melocchi for the support with TCGA data mining and analysis, C Mondello for mouse genomic DNA, J Caceres for the T7-tagged SRSF3 expression vector, V Lemmon and PO Couraud for providing the RaL1cd antibody and hCMEC/D3 cells, respectively, and PP Di Fiore for support. We also thank the patients who generously agreed to donate their surgical samples, and the staff of the Division of Gynecology and of the Biobank of European Institute of Oncology for collecting and annotating the human samples. This work was supported by grants from the Associazione Italiana per la Ricerca sul Cancro (AIRC, projects IG-17395 to CG2, IG-14622 to UC, IG-18683 to ED and IG-18853 to RG, IG-19919 to DG), Worldwide Cancer Research (formerly known as Association for International Cancer Research; AICR 10–0091 to UC), the European Research Council (ERC) under the European Union's Horizon 2020 program (ERC-StG-LS2-637591 to MI) and the Spanish Ministerio de Economía y Competitividad (BFU2017-89201-P to MI). EB, FA and ADM were supported by AIRC - FIRC ITALY postdoctoral fellowships. MG was supported by a fellowship from Fondazione IEO-CCM. Research in FF laboratory is supported by The Giovanni Armenise-Harvard Foundation, and by the Italian Ministry for Education, University and Research (MIUR): Programma Giovani Ricercatori "Rita Levi-Montalcini" and Dipartimenti di Eccellenza Program (2018–2022) - Dept. of Biology and Biotechnology "L. Spallanzani", University of Pavia. AC is supported by a Marie Curie Individual Fellowship from the Horizon 2020 EU Program (Grant agreement n. 745934 – COTETHERS). CG2 is a consultant for Gene Tools, LLC JS and JM are affiliated with Gene Tools, LLC. Funding bodies had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.

Ethics

Human subjects: Fresh tissue samples were obtained upon informed consent from patients undergoing surgery at the Gynecology Division of the European Institute of Oncology (Milan). Sample collection was performed under the protocol number R789-IEO approved by the Ethics Committee of the European Institute of Oncology.

Animal experimentation: All animal studies were performed following a protocol approved by the fully authorized animal facility of European Institute of Oncology and by the Italian Ministry of Health (as required by the Italian Law) (IACUCs number 1256/2015) and in accordance to EU directive 2010/63. Mouse tissues were obtained from Karolinska Institutet (Stockholm, Sweden) and IRCCS San Raffaele Scientific Institute (Milan, Italy), in accordance to Institutional Animal Care and Use Committees.

Senior Editor

  1. Jonathan A Cooper, Fred Hutchinson Cancer Research Center, United States

Reviewing Editor

  1. Douglas L Black, University of California, Los Angeles, United States

Reviewer

  1. Patrick A Murphy, UConn Health, United States

Publication history

  1. Received: December 12, 2018
  2. Accepted: February 8, 2019
  3. Version of Record published: March 4, 2019 (version 1)
  4. Version of Record updated: March 8, 2019 (version 2)

Copyright

© 2019, Angiolini et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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