Triple-negative breast cancer (TNBC) tumours that lack expression of oestrogen, and progesterone receptors, and do not overexpress the HER2 receptor represent the most aggressive breast cancer subtype, which is characterised by the resistance to therapy in frequently relapsing tumours and a high rate of patient mortality. This is likely due to the resistance of slowly proliferating tumour-initiating cells (TICs), and understanding molecular mechanisms that control TICs behaviour is crucial for the development of effective therapeutic approaches. Here, we present our novel findings, indicating that an intrinsically catalytically inactive member of the Eph group of receptor tyrosine kinases, EPHB6, partially suppresses the epithelial–mesenchymal transition in TNBC cells, while also promoting expansion of TICs. Our work reveals that EPHB6 interacts with the GRB2 adapter protein and that its effect on enhancing cell proliferation is mediated by the activation of the RAS-ERK pathway, which allows it to elevate the expression of the TIC-related transcription factor, OCT4. Consistent with this, suppression of either ERK or OCT4 activities blocks EPHB6-induced pro-proliferative responses. In line with its ability to trigger propagation of TICs, EPHB6 accelerates tumour growth, potentiates tumour initiation and increases TIC populations in xenograft models of TNBC. Remarkably, EPHB6 also suppresses tumour drug resistance to DNA-damaging therapy, probably by forcing TICs into a more proliferative, drug-sensitive state. In agreement, patients with higher EPHB6 expression in their tumours have a better chance for recurrence-free survival. These observations describe an entirely new mechanism that governs TNBC and suggest that it may be beneficial to enhance EPHB6 action concurrent with applying a conventional DNA-damaging treatment, as it would decrease drug resistance and improve tumour elimination.
EphA (EPHA1–EPHA8 and EPHA10) and EphB (EPHB1–EPHB4 and EPHB6) receptors comprise the largest group of receptor tyrosine kinases (RTKs) in human tissues. Their ligands, ephrins, are divided into A and B classes based on structural properties: ephrin-As (ephrin-A1–ephrin-A5) are GPI-anchored cell membrane proteins, and ephrin-Bs (ephrin-B1–ephrin-B3) display transmembrane and cytoplasmic domains. Ephrin binding induces tyrosine phosphorylation of Eph receptors, which enhances their catalytic activity and potentiates interactions with cytoplasmic partners, allowing for the control of a complex array of signalling pathways [1, 2]. Interestingly, both EphA and EphB groups possess kinase-deficient members, EPHA10 and EPHB6, suggesting that these molecules may have a crucial role in modulating biological outputs in the Eph receptor network . Through their basal or ligand-induced signalling, kinase-active Eph receptors are frequently implicated in enhancing malignant behaviour of cancer cells  and in controlling tumour-initiating cells (TICs) . In contrast, a strong negative correlation exists between the aggressiveness of solid tumours and kinase-dead EPHB6, with EPHB6 expression frequently reduced in aggressive malignancies, including invasive melanoma , metastatic lung and colorectal cancers , aggressive neuroblastoma [7, 8], prostate, gastric and ovarian tumours [9,10,11]. EPHB6 also suppresses metastasis in xenograft models of human lung cancer , melanoma  and colorectal cancer , while our previous work indicates that it undergoes tyrosine phosphorylation in breast cancer cells and inhibits breast cancer invasiveness . Despite accumulating evidence, suggesting an important tumour-suppressing role for EPHB6, our understanding of its function in malignancy is far from complete. Here, we discuss our novel findings, describing a complex and intriguing action of EPHB6 in controlling the initiation, growth and drug resistance of triple-negative breast cancer (TNBC) tumours that lack the oestrogen receptor (ER), progesterone receptor (PR), do not overexpress the HER2 receptor, and represent the most aggressive breast cancer type .
EPHB6 expression is reduced in breast cancer tumours, but is better preserved in TNBC
While EPHB6 expression is reduced in invasive breast cancer cell lines [17, 18], little is known about EPHB6 behaviour in breast cancer tumours. To fill this knowledge gap, we analysed the TCGA gene expression database, assessing EPHB6 status in 530 tumours and 62 normal samples. Our investigation revealed that EPHB6 abundance is significantly reduced in breast cancer (Fig. 1a), which expanded on previous observations that relied solely on breast cancer cell lines. Unexpectedly, our work with the TCGA and European Bioinformatics Institute (EBI) ArrayExpress datasets  showed that EPHB6 expression negatively correlates with the expression of ER and PR (Fig. 1b, c), suggesting that it might be better maintained in TNBC. Indeed, we found that EPHB6 expression was significantly better preserved in TNBC tumours (Fig. 1d, e) and a similar trend was also observed in breast cancer cell lines, although it did not achieve a statistical significance there, most probably because EPHB6 levels became more variable in the absence of the selective pressure of tumour microenvironment (Supplementary Figure S1A). Taken together, these data implied that EPHB6 may have a prominent role in the biology of TNBC.
EPHB6 suppresses EMT in TNBC cells
To address EPHB6 functions in TNBC, we used TNBC cells, MDA-MB-231, that are highly invasive, have passed epithelial-to-mesenchymal transition (EMT)  and lost EPHB6 expression [17, 18]. EPHB6 expression was restored by transfecting MDA-MB-231 with cDNAs encoding wild-type EPHB6 (MDA-B6) or Myc-tagged EPHB6 (MDA-B6-M) (Fig. 2a), while the empty pcDNA3 expression vector was used as a control (MDA-pc3), as we reported . Our work with these cells revealed that EPHB6 efficiently suppresses their motility (Supplementary Figure S1B). When cultured in individual colonies, MDA-B6 and MDA-B6-M mostly formed very compact colonies with tight cell–cell contacts, while MDA-pc3 cells were predominantly organised in scattered colonies with loose cell–cell interactions (Fig. 2b, c). EPHB6 ability to reduce scattering was not limited to MDA-MB-231, as silencing EPHB6 expression in TNBC cells, BT-20, (Fig. 2d) that are innately EPHB6-positive (Supplementary Figure S1C), increased the formation of scattered colonies (Fig. 2e). Consistent with its apparent role in cell interactions, EPHB6 was frequently found in the areas of cell–cell contact formation in both MDA-B6-M and BT-20 cells and co-localised there with a tight junction protein, ZO-1 (Fig. 2f).
As EMT is associated with increased cell motility and reduced contacts between cancer cells, our observations indicate that EPHB6 could antagonise this process. Indeed, EPHB6 restoration in MDA-MB-231 changed their morphology from irregular to cobblestone-like, typical for epithelial cells (Fig. 3a). This was accompanied by the reduced presence of β-catenin in the nuclei (Fig. 3b) and by the inhibition of transcription-enhancing β-catenin action, which actively supports EMT  (Fig. 3c). These findings correlated with a recent report, showing that EPHB6 reduces β-catenin expression . To further analyse how EPHB6 impinges upon EMT, we evaluated its effect on the expression of an EMT marker, vimentin, which is usually present in mesenchymal cells and promotes this process . Consistent with EMT-suppressing EPHB6 action, vimentin levels proved to be decreased in the presence of EPHB6, while EPHB6 silencing enhanced vimentin expression even in BT-20 cells (Fig. 3d) that had passed EMT .
Collectively, our data suggested an active role for EPHB6 in EMT suppression. In further support of this, our analysis of the TCGA database revealed that EPHB6 expression negatively correlates with that of vimentin in TNBC tumours (Fig. 3e). Interestingly, we did not detect any correlation between EPHB6 and an epithelial marker, E-cadherin, in the TCGA dataset and no significant effect of EPHB6 on E-cadherin expression was evident in our experiments (Fig. 3f, g). These observations indicate that although EPHB6 consistently inhibits EMT-associated behaviour of cancer cells and shifts the balance in favour of the mesenchymal-to-epithelial transition (MET), it achieves only partial EMT suppression. This was further supported by our findings, showing that although EPHB6 does not produce consistent effects on EMT/MET markers, the Met receptor and laminin, it significantly decreases the expression of an EMT-related protein, N-cadherin, in TNBC cells (Supplementary Figure S1D).
The EPHB6 receptor increases tumour-initiating activity
Strong evidence has been presented to support both a model, where EMT favours tumour initiation , and a notion, suggesting that MET promotes TICs generation . Therefore, we examined EPHB6 effect on proliferation of TNBC cells producing tumourspheres, as tumourspheres better represent tumour behaviour than cells cultured in monolayers and are predominantly formed by TICs [27,28,29,30,31,32,33,34]. Analyses of Ki-67 staining and BrdU retention showed that EPHB6 increases cell proliferation in these structures (Fig. 4a, b). The biological relevance of this effect was confirmed by our findings that EPHB6 expression significantly accelerates expansion of tumoursphere cells (Fig. 4c–g, Supplementary Figure S1E and F). Although the effect of EPHB6 silencing was relatively limited in BT-20 in comparison to the effect observed in TNBC cells, HCC70 (Fig. 4d, f) that also express EPHB6 (Supplementary Figure S1C), it was statistically significant and consistently observed (Fig. 4d, Supplementary Figure S1E and F). Overall, these observations indicated that EPHB6 could be involved in the regulation of TIC propagation. Indeed, EPHB6 presence strongly enhanced expression of EpCAM (Fig. 4h), which was previously characterised as breast cancer TIC marker [29, 35]. Moreover, EPHB6 also elevated expression of the OCT4 transcription factor (Fig. 5a) that supports TIC activity [36, 37]. Consistent with its reported function in breast cancer TICs [36, 37], OCT4 silencing strongly reduced the expansion of tumourspheres (Fig. 5b, c), while not decreasing EPHB6 expression (Supplementary Figure S1G). Taken together, this suggested that EPHB6 ability to augment expansion of tumoursphere cells depends on the elevated OCT4 expression.
Since TICs are responsible for tumour initiation and self-renewal, their increased proliferation should result in a higher rate of initial tumour growth. To assess EPHB6 effect on TNBC tumours, we injected MDA-pc3 or MDA-B6-M cells into mammary fat pads of NU(NCr)-Foxn1nu (athymic) and NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NOD-SCID) mice (1.5 × 106 cells and 1.5 × 105 cells per animal, respectively). In both models, mice developed tumours with higher initial growth rates, when injected with EPHB6-expressing cells (Fig. 6a, b). Staining for a blood vessel marker, CD34, showed no difference in vascularisation levels (Fig. 6c), indicating that EPHB6 ability to enhance tumour growth was not because of its effect on neovascularization.
To assess EPHB6 effect on tumour initiation, we injected mice with decreasing doses of cancer cells. This consistently resulted in more frequent development of EPHB6-positive tumours, and statistical analysis confirmed that EPHB6 supports expansion of TIC populations (Fig. 6d). To further validate these observations, we challenged NOD-SCID mice with TNBC cells, HCC70. Consistent with our initial results, silencing of EPHB6 expression in HCC70 decreased tumour growth and tumour initiation, and reduced TIC frequency (Fig. 6e, f). Western blot analyses confirmed that introduced alterations in EPHB6 expression were preserved in both MDA-MB-231 and HCC70 xenograft models (Supplementary Figure S1H). In addition, a similar effect of EPHB6 silencing on tumour growth was observed with TNBC cells, BT-20 (Supplementary Figure S2A). Collectively, these data strongly support the notion that EPHB6 enhances expansion of TIC populations and promotes tumour development. Interestingly, we did not observe any consistent effect of EPHB6 on the CD24loCD44hi combination, identifying some breast cancer TIC populations and the ALDEFLUOR assay has not revealed a consistent effect on another TIC marker, ALDH1. This lack of effect on some TIC markers indicated that in the analysed cell lines, EPHB6 enhanced tumour initiation by expanding a restricted subset of TICs that expressed higher levels of OCT4 and accelerated tumour growth.
EPHB6 effect on TNBC cells is mediated by the Ras-Erk pathway
To gain an understanding of cytoplasmic signalling used by EPHB6 to augment cell proliferation in tumourspheres, we monitored its effect on essential signalling pathways. While we could not detect any consistent effect of EPHB6 on p38, STAT3, mTOR or JNK, EPHB6 enhanced the activating phosphorylation of the ERK1 and ERK2 kinases (Fig. 7a, b, Supplementary Figure S3A). As RTKs typically interact with the GRB2 adaptor protein to activate the RAS-RAF-MEK-ERK pathway , we examined if EPHB6 is also associated with GRB2. Indeed, we consistently observed EPHB6 in GRB2 immunoprecipitates (Fig. 7c). Moreover, EPHB6 expression triggered the activation of the RAS GTPase and RAF kinase (Fig. 7d, e), and EPHB6-induced Erk phosphorylation relied on MEK activity (Fig. 7f), indicating that EPHB6 uses the conventional RAS-MAPK cascade for activating ERK kinases. ERK kinases are known to enhance OCT4 expression , and to promote pluripotency in human cells [39, 40] and therefore could be essential for mediating EPHB6 responses. A crucial role for ERK signalling was confirmed in our experiments, showing that blockage of ERK activation with MEK inhibitors negated EPHB6 effects, reducing OCT4 expression and curbing cell proliferation in tumourspheres (Fig. 7g, h; Supplementary Figure S3B–D). In agreement, ERK2 silencing strongly inhibited EPHB6-triggered proliferative responses and OCT4 expression (Fig. 7i, j; Supplementary Figure S3E–G).
The EPHB6 receptor reduces resistance of TNBC tumours
Increased tumour-initiating activity is expected to result in enhanced drug resistance [41, 42]. To examine if EPHB6 supports cancer drug resistance, we treated mice with MDA-pc3 or MDA-B6-M tumours with a DNA-damaging drug, doxorubicin, which is frequently used in TNBC therapy. To our surprise, growth of MDA-B6-M tumours was strongly inhibited by doxorubicin, while it produced a very small effect on MDA-pc3 tumours (Fig. 8a). Moreover, doxorubicin also efficiently suppressed tumours initiated by HCC70 cells, while EPHB6 silencing enhanced their resistance (Fig. 8b).
The relevance of our findings was further supported by our work with a TNBC patient-derived xenograft (PDX) model, HCI-010, which closely follows tumour behaviour . HCI-010 tumours are EPHB6-positive and there, EPHB6 action mimicked responses that we initially observed in TNBC cell lines. EPHB6 silencing in HCI-010 increased the vimentin level, suppressed OCT4 expression and inhibited expansion of tumoursphere cells (Fig. 9a–e, Supplementary Figure S4A–D). Interestingly, consistent with EPHB6 ability to support tumoursphere expansion and tumour initiation (Fig. 6d, f), its silencing significantly suppressed ALDH1-positive HCI-010 population, which is expected to maintain TIC activity  (Fig. 9f, Supplementary Figure S4E). Reduced EPHB6 expression also decreased ERK activation (Fig. 9g, Supplementary Figure S4F), and cell proliferation in tumourspheres proved to depend on EPHB6-activated ERK signalling (Fig. 9h–j, Supplementary Figure S4G). In NOD-SCID mice, PDX HCI-010 tumours were treated with doxorubicin or saline, as a control. Consistent with EPHB6 action in TNBC cell lines, EPHB6 silencing reduced growth rates in saline-treated tumours, while increasing tumour resistance to doxorubicin (Fig. 10a–c, S5A–C). As in cell lines-based models, EPHB6 silencing was maintained in PDX tumours (Supplementary Figure S4H).
Dormant slow-proliferating TICs are likely to escape cytotoxic therapies targeting fast-proliferating cells and are responsible for cancer relapse [42, 45, 46]. Taken together, our observations suggest an intriguing model, whereby EPHB6 accelerates expansion of TNBC TICs, while also increasing tumour sensitivity to DNA-damaging compounds and potentially other therapies selectively affecting proliferating cells, probably by driving TICs out of their slow-proliferating, resistant state. This model is further supported by our finding that EPHB6 increases killing of tumoursphere cells by therapeutic compounds that depend on high rates of cell proliferation, such as doxorubicin or docetaxel, while not improving elimination of cancer cells in monolayers, where EPHB6 has no significant effect on cell propagation  (Supplementary Figure S6A and B). Moreover, EPHB6 does not increase the sensitivity of tumoursphere cells to bortezomib, a cancer drug that acts as a proteasomal inhibitor and should not directly depend on the proliferative activity (Supplementary Figure S6C). Since TNBC is mostly treated with compounds acting on fast-propagating cells, including doxorubicin and taxane derivatives [48, 49], our model predicts better relapse-free survival of TNBC patients with high EPHB6 expression in their tumours. Consistent with this, our analysis revealed a positive correlation between EPHB6 expression and the long-term recurrence-free survival of breast cancer patients with basal-like tumours that mostly represent TNBC  (Fig. 10d).
TNBC tumours represent the most lethal type of breast cancer because of the high level of drug resistance, high metastasis and lack of targeted therapies . An understanding of mechanisms governing TNBC cells is critical for the development of efficient treatments for this aggressive malignancy. Our findings show that in agreement with its previously reported anti-invasive properties [15, 47], EPHB6 partially reverses the EMT phenotype in TNBC cells. Our observations also indicate that partial EMT suppression induced by EPHB6 is associated with the expansion of tumoursphere cell populations that are mostly represented by TICs [27,28,29,30,31,32,33,34]. Moreover, EPHB6 action also results in the elevated OCT4 expression and in the enlarged ALDH1-positive population in the HCI-010 PDX. Since these factors are characteristic traits of TICs [29, 35,36,37, 44, 51], our data strongly support a model, whereby the EPHB6 receptor concurrently suppresses EMT and promotes proliferation of TNBC TICs. In agreement, EPHB6 augments tumour initiation and expansion of TIC populations in animal models, ultimately confirming the role for this receptor in TIC biology. Nevertheless in established cell lines, MDA-MB-231 and BT-20, EPHB6 presence did not expand CD24loCD44hi or ALDH1-positive subpopulations, selectively increasing EpCAM expresion that represents some breast cancer TICs. This suggests that EPHB6 does not have a blanket effect on TICs, but most likely promotes specific subsets of these cells, and agrees very well with TIC heterogeneity, which results from the evolution of TICs in developing tumours [52, 53]. Our observations also reflect an unfortunate reality that there is no universal marker that defines all types of breast cancer TICs [51, 54] and indirectly suggest that EPHB6 itself may serve as a TIC marker in TNBC.
OCT4 is known to support cell proliferation  and EPHB6-induced proliferation in tumourspheres proved to rely on the increased OCT4 expression. This represents a novel mechanism of the regulation of cell proliferation, as Eph receptors have not been shown to control OCT4 activity. EPHB6 has been previously reported by our team and other groups to activate ERK kinases; however, the relevance of this signalling to cell proliferation and its mechanism have not been addressed [22, 56, 57]. Our investigation reveals that EPHB6 interacts with GRB2 and uses RAS-ERK signalling to elevate OCT4 expression. In agreement, inhibition of the Erk pathway also blocks EPHB6 ability to enhance cell proliferation in tumourspheres. Taken together, our observations support a model, where the EPHB6 receptor acts in TNBC to partially reverse EMT, while its signalling through the RAS-ERK cascade increases OCT4 expression, thus augmenting expansion of TIC populations (Fig. 11). This agrees with a report, demonstrating that the epithelial-like state is associated with a high proliferative activity in breast cancer TICs . This is also consistent with previous observations, showing that ERK2 enhances TIC-like characteristics in immortalised breast epithelial cells .
EPHB6 activity in supporting cell proliferation suggests that it should be beneficial for some TNBC tumours to maintain its expression. Indeed, analyses of the TCGA and EBI databases reveal that although EPHB6 expression is generally reduced in breast cancer, it is better preserved in TNBC tumours. While some of these results differ from the previous report, suggesting that EPHB6 expression may be increased in overall breast cancer , this difference is likely a reflection of the smaller database used in the earlier investigation.
Remarkably, tumours formed by EPHB6-expressing TICs proved to be much more sensitive to treatment with doxorubicin, which could be in part due the ability of EPHB6 to convert TICs into faster proliferating, less resistant cells. Targeting TICs is an important approach in cancer therapy [29, 46, 61] and counterintuitively, our results caution against inhibiting molecules that support TIC proliferation, when using DNA-damaging drugs, as this is likely to drive TICs into a drug-resistant state. Our investigation also indicates that it could be beneficial to support EPHB6 activity in TNBC tumours, when using conventional DNA-damaging treatment, as this would improve tumour elimination, while also suppressing invasive properties of cancer cells.
Taken together, our observations reveal a new molecular mechanism, whereby the EPHB6 receptor controls initiation, growth and drug sensitivity of TNBC tumours, and are likely to have a broad impact on the development of effective therapeutic strategies for this aggressive malignancy.
Materials and methods
Anti-β-tubulin (sc-9104), anti-GRB2 (sc-255), anti-β-catenin (sc-7199), anti-EPHB6 (sc-134332), anti-ERK1/2 (sc-94), anti-ERK2 (sc-154) and anti-Met (sc-10) were from Santa Cruz, anti-EPHB6 (SAB1403784) was from Sigma. Anti-OCT4 was from Cell Signaling Technology (2840) or STEMCELL Technologies (60093). Anti-CD34 (ab81289) and pan-specific anti-laminin (ab7463) were from Abcam. Anti-E-cadherin (610182) and anti-Ki-67 (550609) were from BD Biosciences. Anti-phospho-mTOR (AF1665) was from R&D Systems. Anti-phospho-Erk1/2 (4370), anti-phospho-p38 (4511), anti-phospho-STAT3 (9145), anti-phospho-JNK (4668), anti-EpCAM (2929), anti-GAPDH (2118) and anti-N-cadherin (4061) were from Cell Signaling. Anti-phospho-c-Raf (05–538) was from Millipore.
MDA-MB-231, BT-20 and HCC70 were obtained from ATCC and passaged for less than three months following resuscitations. Therefore, no additional authentication was performed. Mycoplasma testing was performed. For culturing in individual colonies, cells were seeded into 6-well plates (1 × 103 cells per well) and grown for 5–7 days. HCI-010 were previously characterised by one of the authors . Single-cell preparations were made from HCI-010 tumours, as described . HCI-010 cells were cultured in Ultra-Low attachment plates (Corning) using DMEM/F-12 medium, containing B27 supplement (1× Gibco), gentamicin (50 µg/ml; Gibco), hEGF (20 ng/ml; BPS Bioscience), bFGF (20 ng/ml; StemRD), insulin (10 µg/ml, Gibco), hydrocortisone (0.5 µg/ml; STEMCELL) and heparin (2 µg/ml; STEMCELL).
Cells were fixed in 3% paraformaldehyde or 70% ethanol, incubated with indicated antibodies for 40–60 min, rinsed twice (0.5% BSA in PBS), stained with secondary antibodies, and analysed using Beckman Coulter Epics XL (Beckman Coulter Canada, LP., Mississauga, ON, Canada) or Miltenyi Biotec MACSQuant VYB (Miltenyi Biotec Inc. Auburn, CA, USA) flow cytometers and FlowJo software.
Proliferation in tumourspheres
Cells were seeded into 24- or 96-well Ultra-Low attachment plates (4 × 103 or 2 × 103 cells per well, respectively) in complete Mammocult medium (STEMCELL) and allowed to propagate in tumourspheres for 7 days. For each replicate, tumourspheres from 12 independent wells were combined and dissociated using Trypsin-EDTA (Gibco), and proliferation was assessed by cell counting. Images were obtained using EVOS FL Cell Imaging System microscope (Life Technologies).
All protocols were approved by the University of Saskatchewan Animal Research Ethics Board (AREB). Female athymic nude mice (4–6 weeks old) were from Charles River Laboratories. Breeder pairs of NOD SCID mice were from The Jackson Laboratory. Cells were injected in mammary fat pad regions of 4–6-week-old female mice in 100 µl PBS. Tumours were measured using a digital caliper, tumour volume was calculated as: A/2×B2 (A and B are the long and short tumour diameters, respectively). For doxorubicin treatment, all experimental groups initially had similar average tumour sizes, animals were randomly assigned in all other experiments. Experiments were performed in a non-blinded fashion and terminated according to AREB guidelines.
Luciferase reporter assay
MDA-MB-231 were transduced with Cignal Lenti TCF/LEF Luciferase lentiviral particles (Qiagen) (MDA-Luc) and selected with puromycin (10 µg/ml).
Human EPHB6 cDNA was amplified by PCR using the Elongase Enzyme Mix (Life Technologies) and EPHB6-specific primers: 5′-GGGGACAAGTTTGTACAAAAAA
GCAGGCTTCGCGGGCATGGTGTGTAGCCTATGG-3′, and 5′-GGGGACCACTTTGTA
CAAGAAAGCTGGGTCTCAGACCTCCACTGAGCC-3′. PCR products were inserted into attP1 and attP2 sites of the pDONR221 vector using Gateway BP Clonase Enzyme Mix (Life Technologies) to generate the EPHB6 entry clone. EPHB6 cDNA from the entry clone was transferred into attR1 and attR2 sites of the pLenti CMV Hygo DEST plasmid (Addgene) using the Gateway LR Clonase Enzyme Mix. Lentiviral particles were produced by co-transfecting HEK-293T cells with pMD2G, pMDLg/pRRE and pRSV-Rev plasmids, and EPHB6-encoding lentiviral construct. MDA-Luc cells were transduced with EPHB6-encoding lentiviral particles or mock-transduced with empty vector particles in the presence of 10 µg/ml polybrene (Sigma), and selected with 400 µg/ml hygromycin (Life Technologies). Cells were lysed with the lysis buffer (Promega) and the protein concentration was determined by the Bicinchoninic acid method. Protein concentration was adjusted to 1 µg/µl with the lysis buffer and 10 μl aliquots were mixed with 50 µl of the luciferase assay reagent (Promega). The luminescence signal was quantitated with a Luminometer (Glomax 20/20, Promega, Madison, WI, USA)
Stable cell lines
Generation of stable MDA-B6, MDA-B6-M and MDA-pc3 cell lines was described previously . Stable EPHB6 knock-downs were generated using lentiviral particles, encoding EPHB6-targeting shRNA-1 (shB6–1; Santa Cruz, sc-39957-V), according to the manufacturer’s instructions. Cells were transduced in the presence of 10 µg/ml polybrene and selected with puromycin (10 µg/ml; Sigma) for 5 days.
To transduce cells with other shRNA constructs (Santa Cruz: shOCT4-1, sc36123-SH; shErk2-1, sc35335-SH; Sigma: shEPHB6-2 (shB6-2), TRCN0000010677; shOCT4-2 TRCN0000004882; shErk2-2, TRCN0000010040) and previously described GFP or RFP constructs , procedures were performed as described for the Luciferase assay.
For colony visualisation, cells forming colonies on glass coverslips were fixed in 4% formaldehyde, incubated for 1 h in blocking/permeabilization solution (1% BSA, 5% horse serum, 0.1% saponin in PBS) and stained with rhodamine phalloidin. Images were captured with an Olympus FV1000 microscope and Olympus Fluoview software. Channels were split and merged, and image intensity was adjusted where required using ImageJ software. For cell–cell contact analysis, cells were blocked for 1 h in 1% BSA with 5% horse serum in PBS and permeabilized with 0.1% saponin. Cells were incubated with mouse anti-EPHB6 (Santa Cruz) or a matching non-specific IgG for 72 h, rinsed three times with PBS and incubated with anti-mouse Alexa Fluor 594 (Life Technologies, # 8890) in 0.1% BSA in PBS for 1 h. Cells were rinsed and stained with anti-ZO-1 Alexa Fluor 488 or a matching non-specific IgG Alexa Fluor 488 (Thermo Fisher, #339188, #53-4714-80). ProLong Gold antifade with DAPI (Life Technologies) was used as a mounting medium in all confocal microscopy experiments. Cells were visualised with a Carl Zeiss LSM 700 confocal microscope. Z-stack frames were acquired using ZEN 2012 software.
Dataset analysis and code availability
Two microarray gene expression datasets were downloaded: The Cancer Genome Atlas (TCGA; http://tcga-data.nci.nih.gov) and ArrayExpress (http://www.ebi.ac.uk/arrayexpress/) accession E-GEOD-22220 (Buffa Dataset). All available gene expression data were downloaded. Each dataset was analysed separately to avoid problems that could be caused by combining different microarray technologies, measurement types and normalisation techniques. The downloaded TCGA expression data were originally obtained from Agilent G4502A microarrays and measured as a ratio of the amount of expression in the sample to the amount of expression in Stratagene Universal Human Reference RNA. The downloaded EBI expression data were originally obtained from Illumina HumanRef-8 v1 Expression BeadChip microarrays and expressed as log base 2 intensities. To identify a population of TNBC samples, all breast cancer cases in the TCGA and EBI datasets were grouped into three populations. The first population (“ER positive”) included samples with high levels of ER and PR expression. The second population (“HER2 positive”) included cases that expressed high levels of HER2. The third population (“triple-negative”) represented samples with reduced levels of ER, PR and HER2. These populations were visualised in three dimensions (ER, PR and HER2 expression levels) with the R statistical language and environment (http://www.r-project.org), and parameters gradually adjusted, until separation between the populations could no longer be improved. The relevance of these groupings was confirmed by k-means clustering of the datasets using Euclidean distance and k = 3, and comparison of cluster validity indices for the resultant clusters versus our populations. The k-means clustering was performed using built-in functions in R, and the Dunn and Davies–Bouldin indices were calculated using the clv R library (http://cran.r-project.org/package=clv). Both indices indicated that our groupings were superior to the clusters from k-means for both datasets. This allowed us to avoid any loss of relevant data that could potentially be associated with the introduction of arbitrarily selected cutoff values for defining the triple-negative population. EPHB6 expression was assessed in normal breast tissue (where available), in all breast cancer samples combined, in the triple-negative population or in all breast cancer cases excluding the triple-negative population, as indicated in figure legends.
This approach was verified by an additional independent analysis, where TNBC population was defined as the group of samples with the lowest 10% of ER, PR and HER2 expression. Analysis of these populations confirmed all original conclusions. Statistical significance of the differences in EPHB6 expression was determined by Wilcoxon rank-sum tests, since there was no guarantee that the expression levels were normally distributed. Scatter plots were generated and regression analysis was performed to examine correlations between expression of EPHB6 and of other molecules of interest. Spearman correlation and P-values were calculated using standard functions in the R statistical language and environment (http://www.r-project.org). Boxplots and scatterplots were generated with base R graphics functions.
Kaplan–Meier plot was produced for the recurrence-free survival using the online tool KM-plotter (http://kmplot.com/analysis/) and the 2014 version of the database. The lower tertile was used as a cutoff. All other parameters were left as their default options.
For statistical analyses of experimental data, two-tailed Student’s t-test or Mann–Whitney U-test were used, depending on the comparison of variances. The data met test assumptions and all tests were applied appropriately for each dataset. Sample sizes in all experiments, including animal studies, were selected empirically, with initial estimates based on our experience in similar models. Experiments, including animal studies, were done in a non-blinded fashion. The data are presented as mean ± SD. Statistical significance was defined as P < 0.05.
Western blot data were processed using Odyssey, Carestream, Adobe Illustrator and PowerPoint software. Cropping and adjustment of brightness and contrast in Western blot images was done in PowerPoint.
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We thank Chelsea Cunningham for her help with qPCR experiments and Farhad Maleki for assistance with cluster validity analysis. This work was supported by Canadian Breast Cancer Foundation (CBCF) grant # C7003.
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The authors declare that they have no conflict of interest.
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Toosi, B.M., El Zawily, A., Truitt, L. et al. EPHB6 augments both development and drug sensitivity of triple-negative breast cancer tumours. Oncogene 37, 4073–4093 (2018). https://doi.org/10.1038/s41388-018-0228-x
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