Abstract
Basal-like breast cancer (BLBC) is a highly aggressive breast cancer
subtype frequently associated with poor prognosis. Due to the scarcity
of targeted treatment options, conventional cytotoxic chemotherapies
frequently remain the standard of care. Unfortunately, their efficacy
is limited as BLBC malignancies rapidly develop resistant phenotypes.
Using transcriptomic and proteomic approaches in human and murine BLBC
cells, we aimed to elucidate the molecular mechanisms underlying the
acquisition of aggressive and chemotherapy-resistant phenotypes in
these mammary tumors. Specifically, we identified and characterized a
novel short isoform of Roundabout Guidance Receptor 3 (ROBO3s),
upregulated in BLBC in response to chemotherapy and encoding for a
protein variant lacking the transmembrane domain. We established an
important role for the ROBO3s isoform, mediating cancer stem cell
properties by stimulating the Hippo-YAP signaling pathway, and thus
driving resistance of BLBC cells to cytotoxic drugs. By uncovering the
conservation of ROBO3s expression across multiple cancer types, as well
as its association with reduced BLBC-patient survival, we emphasize its
potential as a prognostic marker and identify a novel attractive target
for anti-cancer drug development.
Subject terms: Breast cancer, Cancer stem cells, Oncogenes, Tumour
biomarkers
Introduction
With over 2 million new cases worldwide every year, breast cancer (BC)
is by far the most frequent malignancy in women [[50]1]. Advances in
early detection, classification, and targeted therapies have led to a
tremendous improvement in patient survival in the last decades. Tumors
with limited size and low metastatic load can be surgically removed and
generally correlate with a good prognosis [[51]2]. Despite this,
approximately 10–15% of BC patients develop metastatic tumors within
three years of diagnosis, which is the major cause of death for BC
[[52]3]. Great research efforts over the last decades have led to the
development of targeted therapies such as small molecule inhibitors or
monoclonal antibodies. However, basal-like breast cancer (BLBC) and
triple-negative breast cancers (TNBC), two largely overlapping BC
subtypes, lack the expression of currently druggable molecular targets
[[53]4, [54]5]. The therapeutic options to treat these malignancies
are, therefore, very limited and mostly restricted to conventional
chemotherapies [[55]6]. Additionally, BLBC lesions rapidly develop
resistance and frequently develop metastases [[56]7–[57]9]. These
complications create an urgent need for the development of new
strategies to improve the treatment efficacy for this particular
disease subtype.
We previously developed and characterized WAP-T mice as a model to
study the biology of basal-like mammary carcinoma [[58]10, [59]11].
WAP-T tumor cells are remarkably plastic in vivo and in vitro, which
accurately recapitulates the biology of metastasizing BLBC
[[60]12–[61]15]. Recently, we treated WAP-T tumors with a combination
of Cyclophosphamide/Adriamycin/5-fluorouracil (short CAF) and observed
that a single cycle of this treatment is not able to eradicate the
disease [[62]16]. Notably, recurrences recapitulated the human
situation, showing increased aggressiveness and dissemination
properties, pronounced epithelial-to-mesenchymal transition (EMT), and
cancer stem cell (CSC) traits [[63]17, [64]18].
In the present study, we sought to investigate the mechanisms allowing
BLBCs to escape cytotoxic treatments. Taking advantage of
high-throughput transcriptome analyses, we identified Roundabout
Homolog 3 (Robo3) as one of the most strongly induced genes in WAP-T
cells surviving CAF treatment in vitro and in vivo. ROBO3 is one of the
four members of the Roundabout (ROBO) receptors family that is involved
in axon guidance pathways [[65]19, [66]20]. The binding of ROBO
receptors to their ligands, the SLIT proteins, generally induces a
chemo-repulsive behavior [[67]21]. As a divergent member of its family,
ROBO3 only shows a weak capacity to interact with SLITs [[68]19]. ROBO3
is most highly expressed during development in the central nervous
system, and its expression is maintained in the adult nervous and
sensory organ systems [[69]22, [70]23]. Notably, ROBO3 has a major role
in commissural axon midline crossing, where it controls the switch
between chemo-attraction and chemo-repulsion [[71]24]. Besides their
well-characterized role in the nervous system, members of the SLIT/ROBO
axon guidance pathway have also been implicated in the regulation of
proliferation, homeostasis, and migration processes in various other
organs [[72]25, [73]26]. Moreover, high-throughput genomic analyses by
the International Cancer Genome Consortium (ICGC.org) revealed that
frequent genetic and epigenetic alterations in pancreatic cancer result
in aberrant expression of axon guidance signaling members [[74]27,
[75]28]. However, studies of ROBO/SLIT signaling in cancers are scarce
and the implication of ROBO3 in mammary carcinoma is lacking so far. In
the present study, we describe for the first time a novel ROBO3 short
isoform (ROBO3s) supporting the survival of BLBC cells upon
conventional cytotoxic chemotherapy with a promising potential as a
diagnostic factor or therapeutic target.
Materials and methods
Cell culture
All cell lines were cultivated in the appropriate culture medium
supplemented with 10% FBS, 100 units/mL penicillin and 100 µg/mL
streptomycin at 37 °C and 5% CO[2], as listed in Table S[76]1.
Animal experiments
WAP-T animals were bred under specific pathogen free conditions and
treated according to German regulations for animal experiments
(Niedersächsisches Landesamt für Verbraucherschutz und
Lebensmittelsicherheit, LAVES, authorization 33.19-42502-04-16/1621).
Briefly, 1 × 10^6 H8N8 cells were injected into the right abdominal
mammary gland of 8 to 10 weeks old virgin in WAP-T-NP8 mice. Once
growing tumors reached 500 mm³, animals were treated with one dose of
100 mg/kg body weight cyclophosphamide (Endoxan, Baxter, Deerfield, IL,
US), 5 mg/kg BW doxorubicin (Cell Pharm, Hannover, DE) and 100 mg/kg BW
5-FU (Medac, Wedel, DE). Tumors were dissected at day 6 post-treatment.
Primary TNBC cell culture
The triple-negative breast cancer samples were obtained from
chemotherapy treated patients, with their informed consent, at the
Clinic Braunschweig upon acceptance of the Ärtzekammer Niedersachsen
(authorization Grae/231/2018). Patient biopsies were placed in a
sterile 2 ml tube containing cell culture medium (DMEM:F12 (PanBiotech,
DE) with 20% FBS (Merck, DE), 0.023 U/ml Insulin, 0.5 µg/ml
Hydrocortison and 10 ng/ml hEGF (all from Sigma Aldrich, DE)) at 4 °C
during transport. Upon arrival, biopsies were minced with a sterile
scalpel in 1–3 mm^3 sections and cultured into a 6-well plate. The
experiments were performed on expanded primary tumor cells at passages
lower than p5.
Chemotherapy treatment
Chemotherapeutic drugs were obtained at the pharmacy of the University
Medical Center Göttingen. Treatment concentrations are provided in
Table S[77]2.
Functional assays
Functional assays assessing proliferation and migration of BLBC cells
were performed 24–72 h after siRNA transfection. Detailed descriptions
of the individual assays are available in the supplementary methods.
Protein analysis
Protein extraction and quantification were performed according to
standard protocols. Samples were subsequently analyzed by western blot
or mass spectrometry. Detailed descriptions of both methods including
mass spectrometry data analysis are provided in the supplements. Full
and uncropped western blots are available in the supplemental data.
Gene expression analysis by qRT-PCR
RNA was extracted using QIAzol® (Qiagen) and reverse transcribed into
cDNA with the M-MuLV reverse transcriptase (New England Biolabs).
Relative gene expression was assessed by SYBR green-based qRT-PCR using
a CFX Biorad system (Bio-Rad Laboratories). Detailed qRT-PCR protocol
and primer list are available in the supplemental methods and Table
S[78]4, respectively.
RNA library preparation
RNA samples were prepared using the TruSeq RNA Library Prep Kit v2
(Illumina) according to the Sample Preparation Guide (Illumina).
Detailed protocols are provided in the supplements.
mRNA sequencing analysis
RNA sequencing raw data were processed in the GALAXY environment
[[79]29] provided by the GWDG ([80]https://galaxy.gwdg.de/). Enrichr
([81]http://amp.pharm.mssm.edu/Enrichr/) and Gene Set Enrichment
Analysis (Broad Institute) were used to identify gene signatures
enriched in the different experimental conditions. Detailed information
for sequencing data processing is available in the supplements.
Analysis of publicly available datasets
Publicly available mRNA-seq and ChIP-seq datasets
([82]https://www.ebi.ac.uk/) were processed in the Galaxy environment
provided by the GWDG, as described in supplemental methods and Table
S3. Patient data from the TCGA-BRCA dataset (downloaded at the
[83]https://xenabrowser.net/) were filtered along with the PAM50
basal-like subtype. Detailed data processing steps are described in the
supplemental methods. Bigwig files of normal (GTex) and breast cancer
tissues (TCGA-BRCA) were downloaded using the recount3 tool
([84]http://rna.recount.bio/) [[85]30]. Patient survival analysis for
ROBO3^low and ROBO3^high-expressing BLBC patients in kmplot.com were
performed with following parameters: Subtype: StGallen-basal, systemic
treatments: endocrine therapy-exclude, chemotherapy-any, split patients
by-autoselect best cutoff.
Data visualization, statistical analysis and figures generation
Plots were generated with R (v4.0.2) in the RStudio environment
(v1.1.383) or with Graphpad Prism v.8.0.1. Error bars indicate the
standard error mean (SEM), results of statistical tests are depicted as
the following: *p < 0.05, **p < 0.01, ***p < 0.001.
Reporting Summary
Further information on research design is available in the [86]Nature
Research Reporting Summary linked to this article.
Results
Murine BLBC cells strongly upregulate an uncharacterized short isoform of
Robo3 during conventional chemotherapy survival
To understand the transcriptional changes that occur in BLBC cells
during conventional CAF chemotherapy resistance, we performed
mRNA-sequencing (mRNA-seq) in two previously characterized chemotherapy
naïve cell lines, pG-2 and H8N8, established from WAP-T tumors [[87]12,
[88]15, [89]17]. Differential gene expression analysis identified 782
genes commonly upregulated and 117 genes commonly downregulated (Fig.
S[90]1A). Comparing the 25 most strongly upregulated genes upon CAF
chemotherapy, 7 genes were shared across both cell lines (Fig. [91]1A).
The upregulation of Robo3 particularly drew our attention because of
its dramatic and consistent upregulation across experimental systems,
and its potential implication in cancer cell proliferation and
metastasis [[92]31] (Fig. [93]1A, B). Robo3 was also upregulated upon
CAF treatment of H8N8 cells orthotopically implanted into syngeneic
WAP-T mice, in vivo (Fig. [94]1C).
Fig. 1. Murine BLBC cells strongly upregulate an uncharacterized short
isoform of Robo3 during conventional chemotherapy survival.
[95]Fig. 1
[96]Open in a new tab
A Venn diagram of the top 25 most strongly upregulated genes in pG-2
and H8N8 cell lines after 48 h CAF chemotherapy (log2FC > 1 and padj <
0.05 relative to vehicle treatment). B Log2 fold change (Log2FC) gene
expression volcano plots for pG-2 and H8N8 cell lines treated with 1:32
CAF chemotherapy for 48 h relative to vehicle treatment. Strongly
upregulated Robo3 labeled (blue). Genes exhibiting a significant
regulation with an adjusted p-value (padj) of < 0.05 are highlighted
(red). C Treatment scheme for groups 1 and 2 of WAP-T mice injected
with H8N8 for tumor induction and subsequent CAF chemotherapy treatment
(right panel). Log2FC gene expression volcano plots comparing groups 1
and 2 (left panel). Upregulated Robo3 labeled (blue). Genes exhibiting
a significant regulation with an adjusted padj of < 0.05 are
highlighted (red). D Integrated Genome Browser (IGV) tracks of mRNA-seq
of pG-2 cells treated with CAF or vehicle for 48 h. Reads corresponding
to Robo3 transcription (red) range from exon 23 to exon 28 upon CAF
treatment but are absent in vehicle controls. E Overview of qRT-PCRs
with primers covering the entire Robo3 gene supports the restriction of
Robo3 transcription to exons 23–28 in pG-2 cells after CAF or 5-FU
treatment for 48 h relative to vehicle. F Schematic depiction of
potentially translated (red) and non-translated (grey) Robo3 exons.
Analysis of open reading frames (ORF) shows a possible start codon for
translation with exon 23. ROBO3 peptide sequences identified by mass
spectrometry are mapped against the amino acid sequence (blue).
Canonical and newly predicted ORFs of ROBO3 terminate on an identical
stop codon. The coding sequence for the ROBO3 transmembrane region is
absent from the novel isoform (yellow). G Protein abundance of murine
ROBO3 after CAF treatment in comparison to a control. The area under
the curve of extracted fragment ion chromatograms was summed up and
averaged for six peptides unique for ROBO3s. H Representative crystal
violet stainings of a migration assay (Boyden chamber) on pG-2 cells
treated either with siRobo3 or siRNA control (left panel).
Quantification of migratory cells (area fraction) for each condition
(right panel). E, G, H: Error bars represent mean ± SEM of three
biological replicates, unpaired t-test: *p < 0.05; **p < 0.01;
***p < 0.001.
Surprisingly, the read coverage of Robo3 in the mRNA-seq data
exclusively mapped to a region spanning from exon 23 to 28 (Fig.
[97]1D). This was validated by qRT-PCRs for Robo3 expression, which
consistently only generated a measurable signal in the 3’ end region
spanning from exon 23 to 28 (Fig. [98]1E, Fig. S[99]1B and Table
[100]S4). To exclude the possibility that our mRNA-seq alignment failed
to correctly map 5’-specific Robo3 reads, we compared our data to
publicly available mRNA-seq data of mouse brains. We observed a read
coverage across all exons, confirming the expected expression of
full-length Robo3 in the nervous system (Fig. S[101]1C).
To investigate if the expression of Robo3 short transcript (referred to
as Robo3s in this manuscript) is induced by an alternative promoter, we
evaluated the occupancy changes of histone 3 at lysine 27 actylation
(H3K27ac), marking active regulatory regions, at the Robo3 locus.
Previously published chromatin immunoprecipitation sequencing
(ChIP-seq) data from our group revealed no detectable H3K27ac signal at
the canonical promoter region of the Robo3 gene region in pG-2 cells
(Fig. S[102]1D) [[103]17]. Additionally, the repressive histone mark
H3K27me3 occupied the gene body region spanning from exon 1 to 17,
indicative of active repression of this area via Polycomb Repressive
Complex 2 (PRC2) activity (Fig. S[104]1D). In contrast, the region
encoding Robo3s was negative for H3K27me3 and showed a strong
accumulation of H3K27ac upon CAF treatment. Interestingly, we found
that Robo3s expression was not restricted to the WAP-T mouse model, as
the murine MMTV-Myc mammary carcinomas also showed specific expression
of Robo3s together with a similar epigenetic profile at its gene locus
and a promoter region-specific H3K4me3 peak in the proximity of exon 17
(Fig. S[105]1E). Taken together, our data strongly support the presence
of an alternative promoter region located between exon 17 and 23 that
drives Robo3s expression independently of the full-length transcript
canonical promoter.
The Robo3s transcript presents a potential open reading frame (ORF)
spanning from exons 23 to 28, sharing the same stop-codon with long
Robo3 isoforms, that could give rise to a ROBO3s protein lacking the
transmembrane domain encoded by exon 17 (Fig. [106]1F). We successfully
verified the appearance of a ROBO3s band at the expected size of 28 kDa
upon chemotherapy treatment in pG-2 and H8N8 cells (Fig. S[107]1F, G).
We could not identify any bands in the size range of full-length ROBO3
(150 to 200 kDa), that were specifically upregulated upon chemotherapy.
To confirm the specificity of the putative ROBO3s band, we
simultaneously treated pG-2 cells with CAF and Robo3 siRNA and observed
the disappearance of the ROBO3s band at 28 kDa (Fig. S[108]1H, I). We
then further confirmed the existence of the ROBO3s gene product by mass
spectrometry (MS). We identified 6 peptides corresponding to a 40 %
coverage of the predicted ROBO3s protein sequence in pG-2 cells upon
CAF treatment. In all analyzed samples no peptide matching the
N-terminal domain of the full-length protein could be detected (Figs.
[109]1F, G and S[110]2).
Based on our previous observations, pG-2 cells surviving CAF
chemotherapy treatment showed increased aggressiveness
[[111]16–[112]18]. Therefore, we questioned if Robo3s induction affects
the tumor cell phenotype. First, we ensured that the treatment with
siRNA targeting Robo3 did not influence the proliferation of Robo3s
negative cells (pG-2 neither express full-length Robo3 nor Robo3s under
basal growth conditions). (Fig. S[113]1J). Interestingly, we observed
that challenging pG-2 cells by reducing the concentration of fetal
bovine serum (FBS) induced Robo3s expression (Fig. S[114]1K). As FBS
gradients are commonly utilized as a chemoattractant in Boyden
chamber-based migration assays, we wondered if siRobo3 treatment could
influence the motility of the tumor cells. Indeed, loss of Robo3s
strongly reduced the capacity of pG-2 cells to migrate in vitro (Fig.
[115]1H). Sphere formation assay under low serum concentrations
additionally showed that loss of Robo3s impairs CSC of pG-2 cells (Fig.
S[116]1L). Collectively, we identified a new short isoform of the
murine Robo3 gene giving rise to an approximatively 28 kDa protein and
that likely plays a role in increased tumor cell aggressiveness upon
chemotherapy.
Human BCs express a short ROBO3 variant that supports cell proliferation and
migration in vitro
To determine if Robo3s expression could also be detected in human BC,
we analyzed publicly available mRNA-seq datasets of BC cell lines. We
observed that approximately 50% of the analyzed cell lines markedly
express a short form of ROBO3 (ROBO3s in this manuscript), slightly
longer than the murine counterpart spanning from exon 18 to 28 under
standard growth conditions (Fig. [117]2A). We selected the HCC1806 cell
line to model BLBC in vitro as these cells harbor a medium ROBO3s
expression level (Fig. [118]2A, S[119]3A). mRNA-seq analysis of HCC1806
cells under normal growth conditions confirmed the presence of the
previously identified ROBO3s transcript (Fig. [120]2B). Similar to the
murine cell lines, investigation of epigenetic modifications in HCC1806
publicly available ChIP-seq datasets revealed the presence of an active
alternative promoter located between exons 16 and 18 and characterized
by strong RNA polymerase II (RNA Pol II), H3K4me3, and H3K27ac
occupancy (Fig. S[121]3B). Moreover, like in the mouse, the first 15
exons of the full-length gene were actively repressed via H3K27me3
occupancy. In line, RT-qPCR on two primary TNBC cells samples, as well
as in HCC1806, confirmed the sole presence of the ROBO3s transcript
(Fig. [122]2C).
Fig. 2. Human TNBC cells express a short ROBO3 variant that supports cell
proliferation and migration in vitro.
[123]Fig. 2
[124]Open in a new tab
A Heatmap of ROBO3 expression levels for each exon across 23 human BC
cell lines derived from publicly available mRNA-seq data. Cell lines
are sorted by the expression level of exon 23 based on reads per
kilobase per million mapped reads (RPKM). B Reads coverage (mRNA-seq)
of ROBO3 in HCC1806 cells 72 h after treatment with control or
anti-ROBO3 siRNAs (siCont and siROBO3, respectively). ROBO3 signal was
only detected in a region spanning from exon 17 to exon 28. siROBO3
treatment (red track) efficiently reduced the ROBO3 signal compared to
siCont (grey track). C qRT-PCRs with primer pairs raised against Exons
1-2, Exons 3-4, Exons 5-6, Exons 9-10, and Exons 27-28 of human ROBO3.
Absolute mRNA levels were measured in two primary TNBC cultures
(passage < p5) and in HCC1806 treated with siCont or siROBO3. A signal
was only obtained for the primer pair Exons 27-28. D Heatmap of ROBO3
expression levels (RPKM) for each exon across publicly available
RNA-seq data sets of TBNC patients. E Schematic depiction of
potentially translated (red) and non-translated (grey) ROBO3 exons.
ROBO3 peptide sequences identified by mass spectrometry are mapped
against the amino acid sequence (blue). ORF analysis shows three
possible start codons for translation. Canonical and newly predicted
ORFs of ROBO3 terminate on an identical stop codon. The coding sequence
for the ROBO3 transmembrane region is absent from the novel isoform
(yellow). F, G Protein abundance of human ROBO3 quantified in Jurkat
and HEK293T F as well as MDA-MB-231 and MDA-MB-468 cells G after ROBO3
siRNA knockdown in comparison to a siRNA control. The area under the
curve of extracted fragment ion chromatograms was summed up and
averaged for three peptides unique for ROBO3. F, G: Error bars
represent mean ± SEM of three biological replicates, t-test: *p < 0.05;
***p < 0.001.
We next leveraged publicly available human RNAseq datasets to
characterize expression patterns of ROBO3s in normal and cancerous
tissues. Primary BC lesions showed high ROBO3s expression levels in
more than 50% of the cases in TNBC, BLBC and normal-like subtypes, and
a slightly lower frequency in the other BC subtypes (Fig. [125]2D and
Fig. S[126]3C). Hinting toward a role of ROBO3s upregulation in cancer
in humans, analyses of healthy and cancerous human tissues revealed
that normal cells generally express lower ROBO3s levels (Fig. S[127]3D,
E). Among all analyzed normal human tissues, only the brain cortex
showed a robust expression of the canonical long ROBO3 transcript (GTEx
dataset), further demonstrating that the exclusive ROBO3s detection in
cancerous tissues is not resulting from sequencing artifacts (Fig.
S[128]3F). Interestingly, the expression pattern of normal tissues and
their malignancies were conserved: for instance, liver and
hepatocellular carcinomas almost completely lack ROBO3s expression,
whereas breast and lung, and their respective cancers, showed the
highest expression levels (Fig. S[129]3D, E). Concluding, ROBO3s is the
prevalent ROBO3 transcript variant expressed in normal and cancerous
tissues.
Prediction of potential open reading frames (ORF) in the human ROBO3s
transcript revealed three possible translation start sites, all in
frame with the full-length variant, leading to protein termination at
the canonical ROBO3 stop-codon (Fig. [130]2E). Notably, one predicted
ORF starting at exon 23 presented very high homology with murine ROBO3s
(Fig. S[131]4A). Unfortunately, most commercially available anti-human
ROBO3 antibodies are raised against epitopes of the N-terminal
extracellular domain, rendering the detection of ROBO3s impossible.
Therefore, despite the assessment of different antibodies, western blot
analyses failed to detect ROBO3s at the expected size range (between 18
and 50 kDa, data not shown). Hence, to elucidate whether ROBO3s is
translated into protein in human cancer cells, we again took advantage
of MS analysis. First, as ROBO3s expression is particularly high in
HEK293T and Jurkat cells, we performed a ROBO3s peptides discovery
approach in these cell lines. MS analysis identified three peptides
corresponding to ROBO3s (Fig. [132]2F and S[133]5). We next assessed
the abundance of the identified ROBO3s peptide in the BLBC cell lines
MDA-MB-231 and MDA-MB-468 (Fig. [134]2G and S[135]4B, C). Strikingly,
similar ROBO3s peptides were identified, and their levels were reduced
by siRNA treatment. Here, again, no peptide matching the N-terminal
domains of the full-length protein was detected.
Next, we investigated if ROBO3s has a tumorigenic function in BLBC cell
lines. Knockdown of ROBO3s utilizing two different siRNAs targeting the
short transcript significantly impaired HCC1806 cell proliferation
(Fig. [136]3A, B, S[137]4D, E). ROBO3s silencing also affected other
BLBC cell lines in a ROBO3s-expression dependent manner: high
ROBO3s-expressing MDA-MB-468 cells showed a marked proliferation
reduction, while low ROBO3s-expressing HCC-70 cells were not
significantly affected (Fig. S[138]4F, G). Additionally, ROBO3s
knockdown dramatically altered the morphology of HCC1806 cells,
inducing a switch from an elongated motile shape to a rounder phenotype
typical for less motile cells (Fig. [139]3C). Scratch and Boyden
chamber assays further confirmed that ROBO3s loss heavily impaired
HCC1806 cells motility (Fig. [140]3D, E). In line with these findings,
an analysis of the TCGA-BRCA datasets showed that high ROBO3 expression
(ROBO3s^high, as this was the only detected isoform, see Fig. S[141]3C)
strongly correlates with poor survival outcomes for BLBC patients (Fig.
[142]3F). Together, our data revealed conservation of the ROBO3s
isoform from mice to humans and demonstrated that it contributes to the
oncogenic properties of human BLBC.
Fig. 3. Human ROBO3s is essential for cell proliferation and cell migration.
[143]Fig. 3
[144]Open in a new tab
A Validation of siROBO3 knockdown in HCC1806 cells relative to siRNA
control by qRT-PCR. B Proliferation assay of HCC1806 treated with
siROBO3. Relative confluency was measured by Celigo® and normalized to
day 0. Statistics were performed on the area under the curve (AUC). C
Phase-contrast images of HCC1806 cells treated with siROBO3 or siRNA
control (left panel). Quantification of the percentage of round cells
(right panel). D Scratch assay gaps 0 h and 17 h after seeding. HCC1806
cells were treated with siROBO3 and siRNA control (siCont) respectively
(left panel). Quantification of migratory cells based on relative
filled gap area (right panel). E Boyden chamber based migration assay
of HCC1806 cells treated with siROBO3 or siCont, respectively, and
stained with crystal violet (left panel). A quantification of the
migrated cell number is provided in the right panel. F Kaplan-Meier
curve depicting the survival of BLBC patients (source TCGA-BRCA
dataset) grouped according to ROBO3 expression levels (ROBO3 low n = 45
patients, ROBO3 high n = 95 patients). Log-rank (Mantel-Cox) Test
(p = 0.0049). Error bars represent mean ± SEM. All experiments were
performed in three biological replicates. Unpaired t-test. *p < 0.05;
**p < 0.01; ***p < 0.001.
Loss of ROBO3s impairs actin cytoskeleton structures and increases apoptosis
To understand the molecular mechanisms underlying ROBO3s-mediated
cancer cell aggressiveness, we performed mRNA-seq in HCC1806 cells upon
ROBO3s knockdown. Differential gene expression analysis identified 246
downregulated and 158 upregulated genes in siROBO3 treated cells (Fig.
[145]4A). Gene set enrichment analysis (GSEA) identified a reduction of
signatures specific for canonical ROBO3 function in neurons, suggesting
that ROBO3s could share some functions with the long isoform (Fig.
[146]4B, C). Additionally, the GSEA results suggested that ROBO3s loss
in HCC1806 cells impairs actin regulatory pathways (Fig. [147]4D). To
investigate this further, we performed Phalloidin stainings of actin
fibers in HCC1806 cells after ROBO3 knockdown and observed a
significant reduction of actin-mediated cell protrusions, offering a
possible mechanistic insight into the previously observed tumor cell
migration impairments (Figs. [148]3D, E and [149]4E). Similarly,
ROBO3^high BLBC tumors (TCGA-BRCA dataset) enriched for gene sets
associated with positive regulation of axon extension and actin
cytoskeleton regulation (Fig. S[150]6A). In addition to the altered
cytoskeletal dynamics, GSEA revealed that HCC1806 cells treated with
siROBO3s were significantly enriched for apoptosis specific gene
signatures (Fig. [151]4F). To confirm this finding, we performed
Annexin V staining and observed a strong induction of programmed cell
death in HCC1806 cells upon siROBO3s treatment (Fig. [152]4G).
Together, these findings identify the induction of actin-mediated cell
protrusions and inhibition of apoptosis as two mechanisms through which
ROBO3 contributes to tumor aggressiveness.
Fig. 4. Loss of ROBO3s impairs actin cytoskeleton structures and increases
apoptosis.
[153]Fig. 4
[154]Open in a new tab
A Differentially regulated genes in siROBO3 treated HCC1806 cells
relative to siRNA control. 158 upregulated and 246 downregulated genes
were identified (log2FC ≥ │1│, p-adj < 0.05). Genes exhibiting a
significant regulation with a p-adj < 0.05 are highlighted in red. B–D
Control HCC1806 cells enrich the “KEGG Axon guidance” B, the “GO Neuron
projection guidance” C and the “GO Actin-based cell projection” D gene
sets when compared to siROBO3 treated cell in Gene Set Enrichment
Analysis (GSEA) (mRNA-seq). E Representative pictures of Phalloidin
staining for the actin cytoskeleton in siRNA control and siROBO3
treated HCC1806 cells (left panel). Quantification of the number of
actin-mediated cell protrusions in the respective conditions (right
panel). F GSEA of the mRNA-seq data significantly enriched for the
“Hallmark Apoptosis” gene set in the ROBO3 knockdown condition. G
Annexin V assay by Fluorescence Activated Cell Sorting (FACS) for
HCC1806 treated with siROBO3 or siRNA control, respectively. 1: living
cells. 2: early apoptotic. 3: late apoptotic. 4: necrotic.
Quantification of cell populations (left panel). Distribution of cell
populations and gating (right panel). Error bars represent mean ± SEM
of three biological replicates, unpaired t-test. *p < 0.05; **p < 0.01;
***p < 0.001.
ROBO3s loss impairs the Hippo pathway and sensitizes BLBC cells to
chemotherapy
To uncover the underlying molecular mechanisms, we utilized the Enrichr
pathway enrichment tool ([155]http://amp.pharm.mssm.edu/Enrichr/) and
observed that many genes downregulated in HCC1806 cells upon siROBO3s
treatment were associated with pathways regulating the pluripotency of
stem cells (Fig. [156]5A). To test if ROBO3s is involved in HCC1806
stemness, we performed tumorsphere and colony formation assays upon
ROBO3 knockdown. Strikingly, the number and size of spheres and
colonies was drastically reduced upon ROBO3s loss (Fig. [157]5B and
S[158]6B, C). Furthermore, ROBO3s silencing led to a pronounced
reduction of the ALDH family members (Fig. [159]5C) and of the
CD44^high/CD24^low cell population (Fig. [160]5D), both features
associated with CSC-phenotypes [[161]32]. Further supporting this
finding, ROBO3^high BLBC lesions of the TCGA-BRCA dataset displayed a
significant enrichment of mammary stem cell and EMT signatures in GSEA
(Fig. S[162]6D, E). In addition, the Hippo pathway, a critical pathway
for CSC biology and self-renewal, was impaired upon ROBO3s knockdown,
providing an interesting candidate for understanding the mechanism
through which ROBO3s drives a CSC phenotype [[163]33] (Figs. [164]5A
and S[165]6F). The YAP1 transcription factor (TF), which plays a
pivotal role in Hippo signaling, was strongly downregulated upon ROBO3s
loss (Figs. [166]5E and S[167]6F) [[168]34]. The level of TEAD1, one of
the major YAP1 co-TFs, was also strongly reduced in siROBO3s treated
cells (Fig. [169]5E). We therefore hypothesized that ROBO3s may support
CSC properties by inducing the YAP1 transcriptional program. In support
of this, the stem cell TF SOX2, known to be induced by YAP1, was also
strongly downregulated in siROBO3 treated HCC1806 cells at the protein
level (Fig. [170]5E) [[171]35]. Silencing of both YAP1 and TEAD1
phenocopied the impairment of tumorsphere formation observed earlier
upon ROBO3 knockdown (Figs. [172]5F and S[173]6G, H). Noticeably, TEAD1
knockdown strongly accentuated the proliferation deficiency of siROBO3
treated cancer cells, pointing to sensitization of these cells to
further interferences with the YAP1-signaling (Fig. S[174]6I). YAP1 is
regulated through the LATS1/2-kinases that catalyze its phosphorylation
at serine 127. Consequently, YAP1 nuclear translocation is repressed
and the protein is targeted for proteasome degradation [[175]33].
Strikingly, the stimulation of YAP1 activity by using the LATS1/2
inhibitor TRULI (LATSi) completely restored the tumorsphere-forming
capability of HCC1806 cells (Fig. [176]5G), further implicating YAP1
activity loss in the phenotype of ROBO3-silenced cells. Collectively,
our data support a strong involvement of the ROBO3/YAP1-axis in
promoting CSC features of BLBC cells.
Fig. 5. ROBO3s loss impairs the Hippo pathway and CSC characteristics.
[177]Fig. 5
[178]Open in a new tab
A Pathway enrichment analysis (EnrichR web tool) showing that genes
significantly downregulated upon ROBO3 knockdown are enriched for the
KEGG 2019 signature “Signaling pathways regulating pluripotency of stem
cells” and “Hippo signaling pathway”. B Tumorsphere formation assay of
control and siROBO3 treated HCC1806 cells (left panel). Quantification
of tumorspheres number normalized to the control condition (right
panel). C qRT-PCR showing a decrease of stem cell specific ALDH genes
expression in siROBO3 treated HCC1806 cells. D Measurement of CD24 and
CD44 positive population by fluorescence activated flow cytometry in
HCC1806 cells treated with siCont or siROBO3. E Western blot of HCC1806
cells showing protein levels of SOX2, YAP1 and TEAD1 upon ROBO3
knockdown. F Tumorsphere formation assay of siControl, siYAP1 or
siTEAD1 treated HCC1806 cells (left panel). Quantification of
tumorspheres number normalized to the control condition (right panel).
G Tumorsphere formation assay of siCont and siROBO3 treated HCC1806
cells, without or with LATSi (10 μΜ, left panel). Quantification of
tumorspheres number normalized to the control conditions (right panel).
B, D, E, F and G: Error bars represent mean ± SEM of three biological
replicates, unpaired t-test or chi2-test G. *p < 0.05; **p < 0.01;
***p < 0.001.
Since we originally identified Robo3s induction in murine tumor cells
surviving chemotherapy, and since Hippo signaling has been connected to
drug resistance [[179]36], we hypothesized that ROBO3s expression could
support tumor cell resistance by stimulating YAP1 activity. Notably,
YAP1 was shown to promote the expression of several ABC-transporters
and, thereby, support detoxification processes [[180]37, [181]38].
Indeed, ROBO3s silencing resulted in significantly impaired expression
of multiple ABC transporters involved in the resistance of cancer cells
to numerous chemotherapeutic treatments (Fig. [182]6A) [[183]39]. Based
on these observations, we posit that inhibiting ROBO3s expression
sensitizes BLBC cells to chemotherapy. To test this hypothesis, we
assessed the resistance capacity of HCC1806 cells to CAF and cisplatin
treatment upon ROBO3 knockdown. As expected, combination of siROBO3 and
chemotherapy (CAF or cisplatin, respectively) treatment significantly
reduced the proliferation capacity of the cells compared to the single
treatments (Fig. [184]6C, D and S[185]6J–M). In line, ROBO3s knockdown
strongly sensitized HCC1806 cells to increasing doses of CAF or
cisplatin, pointing to a critical role of ROBO3s in rendering BLBC
cells tolerant to cytotoxic therapies (Fig. [186]6D, E). Accordingly,
ROBO3^low BLBC patients showed significantly better survival rates
after adjuvant chemotherapy than ROBO3^high patients (Fig. [187]6F).
Collectively, our results demonstrate that ROBO3s expression increases
the CSC properties and drug tolerance of BLBC cells by activating the
YAP1 signaling. Therefore, interfering with ROBO3s levels may represent
an attractive strategy to sensitize BLBC to conventional
chemotherapies, offering an opportunity for future targeted therapy
approaches.
Fig. 6. ROBO3s loss sensitizes BLBC cells to chemotherapy.
[188]Fig. 6
[189]Open in a new tab
A qRT-PCR of several ABC transporter genes upon ROBO3 knockdown or
siRNA control treatment of HCC1806. B Proliferation assay of HCC1806
cells, treated with siROBO3, 1:16 CAF or in combination. C
Proliferation assay of HCC1806 cells, treated with siROBO3, 1.25 μΜ
cisplatin or a combination. Relative confluency was measured by Celigo®
and normalized to day 0. Statistical analyses were performed on the
AUC. D, E Dose response assay of siCont or siROBO3 treated HCC1806
cells and increasing doses of CAF or cisplatin. F Patient survival
analysis for ROBO3^low and ROBO3^high-expressing BLBC patients treated
with chemotherapy (source: kmplot.com). A, B, C, D and E: Error bars
represent mean ± SEM of three biological replicates, unpaired t-test.
*p < 0.05; **p < 0.01; ***p < 0.001.
Discussion
We identified ROBO3 as a gene consistently upregulated upon
chemotherapy survival in the basal-like mammary carcinoma WAP-T mouse
model. Studies investigating the role of the ROBO family members have
largely been limited to developmental biology with a particular focus
on neurodevelopmental biology [[190]40]. Despite increasing evidence
implicating the SLIT/ROBO-signaling in oncologic pathways, the
complexity and heterogeneity of ROBO proteins function remain
insufficiently understood [[191]41]. Notably, very recent works have
pointed to the important role of ROBO1 expression in driving oncogenic
properties and cisplatin resistance in bladder and non-small cell lung
cancer, respectively [[192]42, [193]43]. In contrast, Chen and
colleagues demonstrated a tumor-suppressive role of ROBO1 in inhibiting
the proliferation of pancreatic cancer via the YY1-ROBO1-CCNA2-CDK2
axis [[194]44]. Similarly, ROBO2 was shown to act as a tumor suppressor
in pancreatic cancer, while being simultaneously a marker of poor
prognosis in inflammatory BC [[195]45, [196]46]. Together, these
findings highlight how ROBO proteins are increasingly and recurrently
being linked to cancer, however, there is a great need to further
clarify these disparities and investigate the molecular mechanisms
underlying them.
Studies assessing the function of ROBO3 in cancer are very scarce. In
2015, Han et al. showed that ROBO3 promotes pancreatic cancer growth
and metastasis [[197]31]. In contrast, Nakamura and colleagues recently
reported a reduction of ROBO3 levels in invasive malignancies of the
breast when compared with normal tissues and postulated a negative
regulation of metastatic behaviors by Neural EGFL Like 2
(NELL2)/ROBO3-signaling [[198]47]. By identifying a short, previously
uncharacterized isoform of ROBO3 (ROBO3s), our present study
significantly contributes to a better understanding of the
ROBO3-signaling complexity. We have demonstrated that, in mammary
carcinomas, ROBO3s stimulates pathways involved in CSC maintenance,
chemoresistance, and cell migration. ROBO3s lacks extracellular and
transmembrane domains and, therefore, cannot bind extracellular ligands
like NELL2 or SLIT, providing a possible explanation for the
discrepancies with the study of Nakamura et al. The ROBO3s transcript
spans from exons 18 to 28 in human cell lines and exons 23 to 28 in
murine cells, respectively. Despite various studies on ROBO3 expression
in neuronal systems and cancer and reports on several slightly
divergent gene variants, this greatly shortened isoform of ROBO3 has,
to our knowledge, never been described and uncovers a clear gap in our
understanding of ROBO3 function [[199]48–[200]50]. Additionally, no
specific intracellular signaling cascade has been identified downstream
of ROBO3 [[201]23]. This anonymity may explain why ROBO3 has been
largely overlooked in studies of ROBO/SLIT signaling in cancer, and its
discovery, therefore, opens the field to future investigations. A
better comprehension of the ROBO3s-dependent signaling could establish
this factor as an important prognostic factor or even as a new target
for personalized therapies. Exact mechanistic insight into the
intracellular signaling cascade of ROBO3 surpasses the scope of this
study but uncovers an urgent demand for further research.
Analyses on a range of publicly available mRNA-sequencing datasets
revealed that most normal mammary tissues, their respective
malignancies and cell lines expressed ROBO3s, making it the most
broadly expressed ROBO3 isoform. Interestingly, a short screen in other
normal tissues and cancer entities (non-small cell lung cancers,
prostate cancers, pancreatic cancers and liver cancers) confirmed the
sole expression of the ROBO3s variant, suggesting that the importance
of ROBO3s in cancer is likely not limited to BC.
ROBO3s was found upregulated in cancer cells surviving conventional
chemotherapy. Indeed, we demonstrated a clear contribution of ROBO3s to
cancer cell proliferation, migration, and stem cell characteristics.
Stimulation of stem cell transcriptional program confers tumor cells’
resistance to various therapies by increasing detoxifying enzyme
activity, immune tolerance and resistance to programmed cell death
[[202]51–[203]54]. While the function of ROBO3 in stemness has not been
investigated, research on the other family members ROBO1 and ROBO2 has
stressed the involvement of the protein family in progenitor cell
identity. Furthermore, ROBO1 and 2 were shown to control pancreatic
progenitor identity by regulating YAP1 signaling [[204]25]. Consistent
with these findings, our data suggest that ROBO3s regulates stemness
through Hippo-signaling. Upon ROBO3s knockdown, BLBC cells show
impaired levels of Hippo pathway members YAP1 and TEAD1. Consequently,
these cells reduce important stem cell associated features like the
capability to form tumorspheres, high SOX2- and ALDH-genes expression
and prominent CD44^high/CD24^low cell population. Consequently,
expression levels of several ATP-binding cassette transporters (ABC
transporters) are reduced, explaining at least partially the gain of
sensitivity to cytotoxic drugs. In line with our in vitro observations,
high expression of ROBO3s in BLBC patients was associated with stem
cell signatures and poor survival upon adjuvant chemotherapy,
demonstrating the translational relevance of our in vitro functional
studies. Interestingly, sustained YAP1/TEAD signaling is not only
responsible for enhanced stem cell properties, but also strongly
stimulates EMT transcriptional programs, cytoskeleton dynamics, and
therefore correlates with poor patient survival [[205]55–[206]58]. Both
axon guidance and tumor cell motility heavily leverage the actin
cytoskeleton [[207]59, [208]60]. It is therefore attractive to
hypothesize that cancer cells hijack ROBO3s function to promote
metastatic outgrowth.
In summary, this study identified ROBO3s as a factor associated with a
multitude of aggressive tumor characteristics with potential prognostic
value for tumor relapse and chemotherapy resistance.
Supplementary information
[209]Authorship Statements^ (947.8KB, pdf)
[210]41419_2022_5197_MOESM2_ESM.pdf^ (1.7MB, pdf)
Reproducibility Checklist / Reporting Summary
[211]Werner_et_al_Supplements^ (59.4KB, docx)
[212]Full and uncropped western blots^ (532.9KB, pdf)
[213]Figure S1^ (1.6MB, tif)
[214]Figure S2^ (4MB, tif)
[215]Figure S3^ (1.7MB, tif)
[216]Figure S4^ (1.9MB, tif)
[217]Figure S5^ (4.2MB, tif)
[218]Figure S6^ (3.3MB, tif)
Acknowledgements