miR-720 is a downstream target of an ADAM8-induced ERK signaling cascade that promotes the migratory and invasive phenotype of triple-negative breast cancer cells

The ADAM8 antibody (B4068) used for Western blotting was purchased from LifeSpan Biosciences. Antibodies against β-actin (AC-15) and β-tubulin (TUB 2.1), and the ERK inhibitor FR180204 (SML0320) were obtained from Sigma-Aldrich. The antibody to detect specific ERK 1/2 phosphorylated forms (pERK1/2) (9101) was obtained from Cell Signaling Technology. The β1-integrin antibody (552828) and its control isotype matched IgG2A (SC3883) were purchased from BD Biosciences and Santa Cruz Biotechnology, respectively. The anti-ADAM8 antibody MAB10311 and its control isotype-matched IgG1 (MAB002) were from R&D Systems.

The MDA-MB-231 and Hs578T human TNBC cell lines and the human umbilical vein endothelial cell (HUVEC) line were purchased from the American Type Culture Collection (ATCC) and maintained in media recommended by ATCC. Stable clones of ADAM8 short hairpin RNA (shRNA) (shA8-20) and control shRNA (shCtrl-3) MDA-MB-231 cells expressing green fluorescent protein were isolated as described previously [24], and kindly provided by Joerg Bartsch (Philipps University, Marburg, Germany). The identity of the MDA-MB-231 cell line and the shA8-20 and shCtrl-3 MDA-MB-231 clones was authenticated using short tandem repeat analysis (Genetica DNA Laboratories), which showed 100 % identity with the MDA-MB-231 cell line of ATCC. The inflammatory breast cancer cell line SUM-149 was kindly provided by Stephen Ethier (University of Michigan Medical School, Ann Arbor, MI, USA) and maintained as previously described [26]. HEK-293 cells were kindly provided by Nader Rahimi (Boston University School of Medicine, Boston, MA, USA). All cell cultures were confirmed to be free of mycoplasma contamination using polymerase chain reaction (PCR) (VenorGeM Mycoplasma Detection Kit, Sigma). To test for the effects of inhibiting ADAM8 activity, cells were treated either with siADAM8 (see below) or with 20 μg/ml anti-ADAM8 antibody MAB10311, or control isotype IgG1 MAB002. To test for the effects of inhibition of β1-integrin signaling, cells were treated with 10 or 20 μg/ml of either antagonist β1-integrin antibody or control isotype (IgG2A rat). To inhibit ERK signaling, cells were treated with 25 μM FR180204.

The human full-length ADAM8 (hADAM8) cDNA (MGC:134985; Genbank:BC115404.1) and the remnant form ADAM8 cDNA were kindly provided by Joerg Bartsch as described previously [27]. The enzymatically inactive mutant of hADAM8 was prepared as described previously [28]. For transient transfection of shA8-20 cells, cultures were incubated for 48 h in the presence of 4 or 8 μg total DNA in six-well or P60 plates, respectively, with Lipofectamine 2000 (Invitrogen) transfection reagent. For transfection of HEK-293 cells, 1 or 2 μg of total DNA in Lipofectoamine 2000 was used, as indicated. Empty pcDNA3 vector (EV) or pcDNA3.1 myc-his vector (EV-3.1) DNA were used as negative controls.

Transient RNAi-mediated ADAM8 knockdown was performed with the following short interfering RNAs (siRNAs) (Qiagen):

MDA-MB-231 cells were transiently transfected with siADAM8-2 or siCtrl. Total RNA was isolated 72 h after transfection using TRIzol reagent (Invitrogen), according to the manufacturer’s protocol and quantified using a Take3 plate reader (BioTek Synergy HT). We chose 72 h post-transfection for functional assays to decrease effects introduced as a result of the transfection protocol. RNA integrity was assessed using the Agilent 2100 Bioanalyzer (Agilent) and RNA 6000 Nano LabChip kit (5065–4476). RNA with an RNA integrity number (RIN) >9.0 was considered good quality. Array analysis was performed using the TaqMan Array Human MicroRNA A + B cards v3.0 (Life Technologies, 4444913) according to the manufacturer’s protocol. Briefly, 1 μg total RNA was amplified and cDNA prepared using Megaplex Reverse Transcriptase (RT) Human Pool Set v3.0 primers (Life Technologies, 4444745) and TaqMan microRNA reverse transcription kit (Invitrogen, 4366596). Subsequently, 6 μl of the cDNA preparation was diluted with TaqMan Universal PCR Master Mix (450 μl) and nuclease free water (444 μl) and loaded on the 384-well TaqMan low-density array card. The cards were then centrifuged to distribute the cDNA samples in the reaction wells using a refrigerated Sorvall and Heraeus bucket centrifuge at 1200 rpm (331 × g) for two 1-min runs. The plate was then sealed and the real-time PCR was carried out on an Applied Biosystems 7900HT Real-Time PCR system. The data were analyzed using SDS software. Relative miRNA expression was calculated by comparing MDA-MB-231 cells with ADAM8 knockdown to cells with the control siRNA. This experiment was performed in duplicate and miRNAs that showed greater than twofold change in both replicates were selected for study.

All animal work was performed in accordance with a protocol approved by the Institutional Animal Care and Use Committee of Tufts University and Tufts Medical Center. Blood was collected from 6-week-old female nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice (Jackson Laboratory) using submandibular bleeding and plasma isolated and processed as described below. The next day, 2.5 × 10⁶ shCtrl-3 MDA-MB-231 cells in 40 μl 50 % Matrigel (BD Biosciences, CB-40230A) solution (1:1 dilution of Matrigel with DMEM medium) were implanted in the fourth inguinal mammary fat pad. Primary tumor growth was monitored by caliper measurement twice a week. Tumor volumes were calculated as (length × width²)/2. At the indicated times, blood was collected and processed as above. Mice were sacrificed after 3–4 weeks when tumors derived from shCtrl-3 cells had reached a volume of ~1 cm³. Tumors were dissected, photographed, weighed and flash frozen.

RNA was isolated from cells using TRIzol reagent and DNA prepared using TaqMan MicroRNA Reverse Transcription Kit (Invitrogen, 4366596) according to the manufacturer’s protocol. Expression of miRNAs was assessed by quantitative reverse transcription PCR (RT-qPCR), using U6 snRNA (Invitrogen, 4427975) as the control. Single tube TaqMan assays (Invitrogen, 4427975) were obtained for all miRNAs of interest (hsa-miR-30d*, hsa-miR-181a-2*, hsa-miR-29c, rno-miR-29C*, hsa-miR-93*, hsa-miR-520c-3p, hsa-miR-130b*, hsa-miR-720, hsa-miR-106*b, hsa-miR-98, and hsa-miR-20a*) and qPCR was carried out as follows: 95 °C for 10 min, then 40 cycles of 95 °C for 15 s and 60 °C for 60 s. All analyses were performed in triplicate and the data were normalized to U6 snRNA. Average fold-change ± SD in miRNA levels relative to those in control untreated cells (set to 1) are presented.

Following isolation of mouse blood via submandibular bleeding, 25 μl of a 10-mM EDTA solution was added to individual samples to prevent coagulation. The samples were stored on ice and centrifuged at 1300 g for 20 min at 4 °C. Supernatants were collected and lysed as recommended in the manufacturer’s protocol for the miRneasy serum/plasma kit (Qiagen, 217184). Subsequently, 3.5 μl of C. elegans miR-39 (Qiagen, 219610) (160 nM) was added as a spike in each sample to control for miRNA recovery. RNA was then isolated as per protocol. Reverse transcription was carried out using the miScript II RT kit (Qiagen, 218161). miRNA expression was assessed by qPCR, and values normalized to the control C. elegans miR-39 (Ce_miR-39_1, MS00019789, Qiagen). The miScript primer assay was used for qPCR of miR-720 (Hs_miR720_1, MS00014833, Qiagen) as follows: 95 °C for 15 min, then 45 cycles of 94 °C for 15 s, 55 °C for 30 s and 70 °C for 30 s. Average fold-change ± SD in normalized miR-720 miRNA levels relative to those in control untreated mice are presented.

Exosome isolation was performed essentially as published previously [29]. Briefly, cell lines were cultured in ten P100 plates each until they reached 50–70 % confluency. The culture media for MDA-MB-231, shA8-20 and shCtrl-3 cells were then replaced with serum-free media, whereas, for SUM-149 cells which require serum for their viability, exosome-depleted FBS media was used. After 72 h, supernatants were collected, centrifuged at 2000 × g for 20 min to remove debris and filtered using a 0.22-μm filter. Exosomes were isolated by ultracentrifugation at 100,000 × g for 70 min. The exosome pellet was washed with PBS and RNA isolated using the miRCURY RNA isolation kit-cell and plant (Exiqon, 300110). Isolated RNA was subjected to Agilent 2100 Bioanalyzer (Agilent) analysis using a RNA 6000 Nano LabChip kit (5065–4476), which confirmed the lack of rRNA in the miRNA samples. Levels of miR-720 expression, determined by RT-qPCR, are presented ± SEM from two independent experiments.

MDA-MB-231 and SUM-149 cells were transfected in six-well plates with 50 nM miR-720 mirVana miRNA inhibitor (Invitrogen, 4464084 (hsa_miR_720, assay id: MH13574)) or negative-control (Invitrogen, 4464076) oligonucleotides using Lipofectamine RNAi Max (Invitrogen). After 16 h, fresh media was added and the cells were collected 48 h after transfection for functional analysis. To overexpress miR-720, ADAM8 knockdown MDA-MB-231 cells (shA8-20 clone) were plated a day prior to transfection. When cells reached 80 % confluency, cultures were transfected overnight with 200 pmol miR-720-mimic (Invitrogen, 4464066 (assay id: MC13574)) or the negative control mimic (Invitrogen, 4464058) using Lipofectamine 2000. The transfection media were replaced with fresh media and cells harvested 48 h after transfection for functional analysis.

Whole-cell extracts (WCEs) were prepared using RIPA buffer (50 mM Tris pH 7.6, 150 mM NaCl, 1 % NP-40, 0.1 % SDS, 5 mM EDTA, 1 % sodium sarkosyl) supplemented with protease and phosphatase inhibitors [30], and 5 mM EDTA and 10 mM phenanthroline to inhibit the autocatalytic activity of ADAM8. Lysates were sonicated and centrifuged at 16,000 g for 15 min. Protein concentration in the supernatants was determined using DC Protein Assay Reagent (BioRad). Samples (25 μg) were subjected to immunoblotting as previously described [27]. Either β-actin or β-tubulin was used as the loading control.

As a measure of cellular metabolism and therefore cell growth, ATP levels were assessed using an ATPlite luminescence ATP detection assay system (Perkin Elmer), as described previously [31]. Briefly, cells (3000 cells/100 μl for MDA-MB-231 and shA8-20, or 8000 cells/100 μl for SUM-149) were plated in 96-well plates. After 24 h, an equal volume of APTlite 1Step luminescence reagent was added and luciferase activity measured. Average ATP levels, for triplicate samples, are presented relative to the control condition set to 1 (± SD).

Matrigel outgrowth assays were carried out as described previously [32], using 5 × 10³ cells plated in duplicate in 24-well plates. Cultures were incubated for 11–15 days and photographed at 10× magnification. Migration and invasion assays were performed in triplicate using polycarbonate filter Transwells (Costar) with 8-μm diameter pores, without precoating or precoated with growth factor-reduced Matrigel (BD Biosciences, 356231), respectively. For transendothelial migration, Transwells were coated with a confluent layer of HUVECs instead of Matrigel. Suspensions of 1 × 10⁵ tumor cells were layered in the upper compartment of the Transwells and incubated at 37 °C. After 24 h, cells that migrated or invaded to the lower side of the filter were quantified by crystal violet staining and OD_570nm determination. The negative control or control mimic condition was set to 100 % and the mean ± SD from three independent experiments is given.

All clinical investigation was conducted in accordance with the principles outlined in the Declaration of Helsinki. Serum samples used in this work were provided by the Institut Régional du Cancer Nantes-Atlantique tumor bank, funded by the Institut National du Cancer (INCa) and approved by the French Minister of higher education and research (n°. AC-2008-141). Informed consent was obtained from patients to use their surgical specimens, serum, and clinicopathological data for research purposes, as required by the French Committee for the Protection of Human Subjects (CCPPRB). Ouest IV – Nantes CCPPRB approved use of serum samples for this study (6 May 2013: n°. 357/2013). This study did not need additional ethical approval.

Blood samples were collected in BD Vacutainer red-top tubes (BD Biosciences, 367837) from 37 consenting women diagnosed with TNBC in the Nantes Cancer Center (ICO, France). At the time of sampling, patients had not received any treatment in the form of surgery, chemotherapy, radiation, or endocrine therapy. Blood samples were allowed to clot for 1 h and centrifuged at 400 g for 10 min. Sera were stored at –80 °C within 2 h of being taken. Detailed patient clinicopathological characteristics are listed in Additional file 1 (Table S1). Sera were also collected from 15 consenting healthy female individuals. ADAM8 protein was measured in the serum using an enzyme-linked immunosorbent assay (ELISA) (R&D Systems) according to the manufacturer’s instructions.

Serum samples from patients (150 μl) were thawed to room temperature and processed as recommended for the NucleoSpin miRNA plasma kit (Invitrogen, 740981). Subsequently, 3.5 μl C. elegans miR-39 (Qiagen, 219610) (160 nM) was added as a spike in each sample to control for miRNA recovery. RNA was then isolated as per protocol. Reverse transcription was carried out using the TaqMan MicroRNA Reverse Transcription Kit (Invitrogen, 4366596). miRNA expression was assessed by qPCR, and values were calibrated to the control C. elegans miR-39 (Cel-miR-39-3p). Single tube TaqMan assays (Invitrogen, 4427975) for hsa-miR-720 were carried out as follows: 95 °C for 10 min, then 40 cycles of 95 °C for 15 s and 60 °C for 60 s. All analyses were performed in triplicate and the data were normalized to hsa-miR-16 (Invitrogen, 4427975). Serum levels of miR-720 in patients versus healthy individuals are presented as average fold-change ± SD.

ADAM8 is synthesized as a multidomain proteolytically inactive proform protein (120-kDa), which can autocatalytically clip its prodomain, leaving an active 90-kDa protein with MP activity, which can be further processed to a 60-kDa remnant transmembrane form lacking the MP domain but retaining the disintegrin domain and C-terminal region (Fig. 1a). As ADAM8 promotes the migratory and invasive phenotype of TNBC cells [24], we sought to determine whether any of these effects were mediated via downstream miRNAs. Firstly, we asked whether ADAM8 regulates miRNA expression in MDA-MB-231 TNBC cells using an RT-qPCR-based miRNA array. Seventy-two hours following transfection of MDA-MB-231 cells with either siADAM8-1 or siADAM8-2 or a control siRNA (siCtrl), WCEs were isolated and subjected to Western blotting (Fig. 1b). Both ADAM8 siRNAs effectively reduced the levels of ADAM8 compared to the siCtrl. As siADAM8-2 was previously shown to be slightly more effective in downregulating ADAM8 function in MDA-MB-231 cells [24], it was selected to knockdown ADAM8 for the miRNA array assay. RNA was isolated from two independent cultures of MDA-MB-231 cells 72 h post-transfection with siADAM8-2. The RNA preparations were subjected to TaqMan low-density miRNA array card analysis. The relative miRNA expression in the siADAM8 RNA preparations compared to the siCtrl RNA was calculated using the SDS software. A threshold increase or decrease of twofold was used as the cut-off, and 68 miRNAs were differentially regulated greater than or equal to twofold upon ADAM8 knockdown with siADAM8-2 (Fig. 1c). Of these 68 miRNAs, 66 miRNAs were downregulated and 2 miRNAs were upregulated. Literature analysis was performed to identify the miRNAs in this subset that had been reported aberrantly expressed in cancer [33‐42], and 11 oncogenic miRNAs (oncomiRs) were identified. To confirm that these miRNAs were indeed regulated by ADAM8, RT-qPCR for these 11 miRNAs was performed using three independently isolated RNA preparations from MDA-MB-231 cells treated with siADAM8-1 or siADAM8-2 versus siCtrl. As seen in Fig. 1d, knockdown of ADAM8 with siADAM8-2 led to reduced levels of all 11 RNAs, confirming the TaqMan low-density miRNA array card analysis. However, treatment with siADAM8-1 led to reduced levels of only eight miRNAs (miR-181a-2, miR-29c, miR-29c*, miR-98, miR-520c-3p, miR-93, miR-130b, and miR-720), whereas three miRNAs showed increased expression, including miR-30d, miR-20a and miR-106*b (Fig. 1d), suggesting these miRNAs may not be regulated specifically by ADAM8 or may have differential regulation via splice variants. Overall, multiple miRNAs appear to be regulated by ADAM8 or a downstream mediator/pathway.

While all of the ADAM8-regulated miRNAs have been implicated in various aspects of tumorigenesis, we were particularly interested in miR-720 since it has been reported to be highly expressed and abundantly released from breast cancer cells [25, 43‐45] and detected in the blood of patients with multiple myeloma [46]. Also, we found that miR-720 was highly expressed in MDA-MB-231 cells (Additional file 2: Figure S1). The transfection of MDA-MB-231 cells with siADAM8-2 and siADAM8-1 led to a 3.7-fold and 1.7-fold downregulation of miR-720, respectively (Fig. 1d). Recently, Lerebours et al. [25] identified miR-720 in a set of five miRNAs as a predictive marker of poor prognosis in patients with inflammatory breast cancer (IBC), which we have found also frequently express ADAM8 (data not shown). Thus, we next analyzed the effects of ADAM8 knockdown on SUM-149 cells, a triple-negative IBC line which has been characterized as a basal-like 2 (BL2) subtype line of TNBC [47] that expresses high levels of ADAM8 [24]. SUM-149 cells were transfected with siCtrl, siADAM8-1 or siADAM8-2, and after 72 h WCEs and RNA were isolated. Western blot analysis confirmed the effective knockdown of ADAM8 by the two specific siRNAs (Fig. 1e). RT-qPCR of RNA isolated in three independent experiments indicated miR-720 levels were reduced by 2-fold and 1.7-fold, respectively, in SUM-149 cells treated with siADAM8-2 and siADAM8-1 compared to the siCtrl (Fig. 1f). Thus, knockdown of ADAM8 decreases miR-720 levels in both MDA-MB-231 and SUM-149 TNBC cells.

We next sought to confirm that miR-720 is secreted from breast cancer cells, as reported previously, and that its secretion is regulated by ADAM8. To address this question, we isolated exosomes released into the media by MDA-MB-231 and SUM-149 TNBC cells, extracted miRNA and subjected the RNA to RT-qPCR for miR-720. Consistent with the findings of Pigati et al. [45], these two breast cancer cells lines were shown to release miR-720 (Fig. 2a). To confirm that miR-720 release is controlled by ADAM8, exosomal levels of miR-720 in cultures of MDA-MB-231 cells with shCtrl RNA (shCtrl-3) versus shA8-20 with stable shRNA knockdown of ADAM8 [24] were compared. The release of miR-720 into the exosomes by shA8-20 cells was decreased relative to the shCtrl-3 cells (Fig. 2b). Thus, TNBC cells release miR-720 in an ADAM8-dependent manner.

To further assess the role of ADAM8 in regulation of miR-720, we ectopically expressed full-length ADAM8 in cell lines that either express low levels of ADAM8 or are ADAM8-negative. In particular, MDA-MB-231 cells with stable ADAM8 shRNA knockdown (shA8-20) and HEK-293 that do not express ADAM8 [24] were selected. WCEs and RNA were isolated 48 h after transfection with an ADAM8 expression vector or with empty vector (EV) DNA as control. Western blotting confirmed the ectopic expression and processing of ADAM8 in these cells (Fig. 2c, d). As judged by RT-qPCR, ADAM8 expression led to an increase in miR-720 level of 1.4-fold in shA8-20 cells (Fig. 2e) and of threefold in HEK-293 cells (Fig. 2f). This was consistent with the relative levels of ectopic ADAM8 overexpression and transfection efficiencies of these lines. Previously, we showed that culturing Hs578T TNBC cells in suspension (3D condition) increased the levels and processing of ADAM8 compared to growth on plastic (two-dimensional (2D) condition) [24]. Thus, we asked whether this increase in endogenous ADAM8 expression affects miR-720 levels. As seen previously, significant increases in ADAM8 levels were detected when Hs578T cells were grown in suspension compared to those grown on plastic (Fig. 2g). RT-qPCR analysis of RNA collected from cells after 48 h of culture revealed that miR-720 levels were elevated more than threefold in Hs578T cells in 3D versus 2D cultures (Fig. 2h). Thus, endogenous or ectopic expression of ADAM8 leads to increased miR-720 levels.

To determine how ADAM8 regulates miR-720 expression, we first tested the role of its MP domain using vectors that express either an ADAM8 protein that is catalytically inactive or lacking the MP domain. Previously, it has been reported that a Glu (E330) to Gln (Q330) mutation (EQ mutation) in the catalytic MP domain of mouse ADAM8 led to a proteolytically inactive protein [22]. We demonstrated that human ADAM8 with analogous EQ mutation was similarly inactive, as judged by its inability to shed a 29-kDa CD23 fragment following co-transfection into HEK-293 cells [28]. Here, HEK-293 cells were transfected with vectors expressing either WT ADAM8 (WT-3.1) or EQ ADAM8 (EQ-3.1) or empty pcDNA3.1 myc/his EV-3.1 vector DNA as control. Western blot analysis confirmed the expression of WT and EQ mutant ADAM8 protein (Fig. 3a). As found previously [28], a band was seen with EQ-3.1 that ran somewhat faster than the 90-kDa active ADAM8 seen with the WT-3.1 vector, presumably resulting from non-autocatalytic proteolysis of the EQ mutant proform. An essentially equal increase in miR-720 levels was triggered by the WT-3.1 and EQ-3.1 ADAM8 proteins (Fig. 3b), suggesting that the activity of the MP domain of ADAM8 is not required for miR-720 induction. To confirm that the metalloproteinase activity was not essential, we tested the ability of the ADAM8 remnant form, which lacks the MP domain, to induce miR-720. HEK-293 cells were transfected with pcDNA3 vectors expressing either WT ADAM8 or its remnant form, or control EV pcDNA3 DNA for 24 or 48 h. Western blot analysis confirmed substantial expression of the remnant form (Fig. 3c). At 24 h, the vectors expressing either the WT or remnant forms of ADAM8 induced the expression of miR-720 equally (approximately threefold each) compared to the EV control condition (Fig. 3d). At 48 h, a further increase in miR-720 levels was observed with both forms, but was more pronounced with the WT likely due to expression of both active and remnant ADAM8 (Fig. 3d). Thus, the remnant form of ADAM8, which lacks metalloproteinase activity, effectively induces miR-720 expression, suggesting the MP domain of ADAM8 is not involved in the induction.

The MP and DI domains have been shown to constitute two independent functional cores of ADAM8 activity [18]. The DI domain and the CRD/ELD portions of ADAM proteins have been suggested to form an essential binding domain [48] for interaction with integrins [49, 50]. Since we have shown that ADAM8 is required for β1-integrin activation [24], which is known to participate in adhesion of breast cancer cells to the endothelium [51, 52], we investigated the potential role of the DI/CRD/ELD domains in miR-720 activation. The effects of treatment with the MAB10311 monoclonal antibody that specifically recognizes the CRD/ELD region of ADAM8 on miR-720 expression were tested. HEK-293 cells ectopically expressing the remnant form of ADAM8 or EV DNA were incubated with 20 μg/ml of either ADAM8 antibody MAB10311 or isotype-matched control IgG1 (MAB002). After 24 h of treatment, RNA was extracted and RT-qPCR analysis performed to measure miR-720 levels (Fig. 3e). The induction of miR-720 by the remnant form was significantly inhibited (~40 % inhibition) upon addition of the anti-ADAM8 CRD/ELD antibody. To determine whether direct inhibition of β1-integrin signaling was sufficient to reduce the levels of miR-720 expression, MDA-MB-231 cells were treated for 24 h with 10 or 20 μg/ml of an antagonist anti-human β1-integrin antibody or with an isotype-matched IgG2A. The levels of miR-720 were significantly decreased by approximately 10-fold upon treatment with the β1-integrin antibody when compared to the control (Fig. 3f). Altogether, these findings indicate that the DI/CRD/ELD domains of ADAM8 play a key role in the regulation of miR-720 via activation of the β1-integrin signaling pathway.

Overexpression of ADAM8 has been shown to also activate ERK/MAPK signaling in osteoclasts [53]. To determine whether ADAM8 leads to ERK activation in breast cancer cells, we tested ERK1/2 phosphorylation (p44/p42) levels in Hs578T cells that overexpress ADAM8 naturally when cultured under 3D conditions versus 2D, as shown above in Fig. 2g. WCEs from Hs578T cells cultured in 2D versus 3D were subjected to Western blotting. In cells grown under 3D conditions, where cells display elevated expression and processing of ADAM8 (Fig. 2g), we observed a 2.7-fold increase in the levels of pERK1/2 (Fig. 4a). A similar increase in levels of pERK1/2 was observed in HEK cells transfected with either WT or the remnant form of ADAM8 (Fig. 4b), indicating that the MP domain is not required for activation of ERK signaling, consistent with the induction of miR-720 observed above. Interestingly, β1-integrin has also been shown to signal predominantly through the recruitment and activation of Src-family kinases, which then activate a downstream cascade including ERK/MAPK and JNK kinases [54]. As phosphorylation of ERK has been implicated in the induction of both cell migration and anchorage-independent growth in breast cancer [55], we asked whether ERK signaling was involved in miR-720 regulation. To address this question, MDA-MB-231 cells were treated with either 25 μM of the ERK specific inhibitor FR180204 [56], or an equivalent volume of DMSO. After 24 h, WCEs and RNA were isolated. As expected, treatment with FR180204 caused a twofold decrease in pERK1/2 levels in comparison to DMSO (Fig. 4c). Notably, a significant decrease in miR-720 expression was observed upon ERK inhibition (Fig. 4d). Similarly, treatment of SUM-149 cells with FR180204 caused a 40 % drop in miR-720 expression (Fig. 4e). Thus, the induction of miR-720 by ADAM8 is mediated via activation of a β1-integrin to ERK1/2 signaling pathway in TNBC cells.

As ADAM8 promotes the migratory and invasive phenotype of TNBC cells [24], the role of miR-720 as a mediator of these aggressive properties was tested using a specific anti-miR. MDA-MB-231 cells were transfected with 50 nM anti-miR-720 for 48 h and a 70 % decrease in miR-720 levels was measured (Fig. 5a), leading to a 40 % decrease in MDA-MB-231 cell migration in a Boyden chamber assay (Fig. 5b). Furthermore, miR-720 knockdown robustly inhibited the capacity of MDA-MB-231 cells to form characteristic branching colonies in Matrigel (Fig. 5c, left panel). The numbers of branched colonies were counted and a ~60 % reduction was seen in three independent experiments (Fig. 5c, right panel). To determine whether miR-720 was likely playing a role in β1-integrin-mediated transmigration of cancer cells through the endothelial layer of blood vessel walls [57], the effects of miR-720 knockdown on transendothelial migration were monitored. MDA-MB-231 cells that had been treated with 50 nM anti-miR-720 for 48 h were plated over a HUVEC monolayer in a Boyden chamber and the number of cells that migrated through the endothelial barrier measured after 24 h. The capacity of MDA-MB-231 cells to transmigrate through the endothelial cell layer was reduced by 60 % after miR-720 knockdown (Fig. 5d). In contrast, knockdown of miR-720 over a 24-h period did not decrease 2D growth of MDA-MB-231 cells (Fig. 5e), consistent with our previous findings that growth of TNBC cells on plastic was unaffected by knockdown of ADAM8 [24]. To extend these observations to a second TNBC line, the roles of miR-720 in SUM-149 cells were evaluated. Knockdown of miR-720 (Fig. 6a) resulted in a 40 % decrease in the ability of SUM-149 cells to migrate (Fig. 6b) and to invade through Matrigel (Fig. 6c), while 2D growth was not affected (Fig. 6d).

Finally, to test whether addition of miR-720 was sufficient to overcome the loss of the aggressive phenotype of TNBC cells upon ADAM8 depletion, cultures of shA8-20 MDA-MB-231 cells stably expressing an ADAM8 shRNA were transfected with 200 pmol of a miR-720 mimic or control miRNA. Expression of the miR-720 mimic resulted in a 10-fold increase in miR-720 levels compared to the control (Fig. 7a). Notably, miR-720 expression was sufficient to profoundly increase the formation of branched colonies from shA8-20 MDA-MB-231 cells in a Matrigel 3D-outgrowth assay (Fig. 7b), and led to more than a 50 % increase in their ability to migrate (Fig. 7c). As expected, growth of the shA8-20 cells on plastic was unaffected by expression of the miR-720 mimic (Fig. 7d). Thus, the ADAM8 downstream target miR-720 participates in many aspects of the invasive and migratory properties of TNBC cells, suggesting that miR-720 is an important mediator of the aggressive properties of TNBC cells induced by ADAM8.

To begin to evaluate the potential of using miR-720 as a biomarker for ADAM8-expressing TNBCs, an orthotopic mouse model was performed using control MDA-MB-231 cells (shCtrl-3 clone), which express high levels of ADAM8 and miR-720. In our preliminary analysis, the plasma levels of miR-720 appeared to vary between individual mice (data not shown). Thus, plasma was collected from each 6-week-old female NOD/SCID mouse (n = 5) a day before TNBC cell inoculation into the mammary fat pad, and used to establish baseline miR-720 levels for each individual mouse. On day 1, 2.5 × 10⁶ shCtrl-3 MDA-MB-231 cells were implanted into the fourth inguinal mammary fat pad and primary tumor growth was monitored by palpation and caliper measurement twice a week thereafter (Fig. 8a). Little growth of the tumors was detected within the first 7–9 days, as reported previously [24]. Plasma was collected 7 days after cell implantation when the tumors were barely palpable. Tumor growth was followed for a total of 21 days (Fig. 8a). A significant increase in miR-720 levels of about 40-fold was detected in the blood of mice at the 7-day time point (Fig. 8b) and remained elevated above 10-fold to day 21 (data not shown). It was interesting to note that the mice with the two fastest growing tumors (mouse 1 and mouse 2) had the highest miR-720 levels at day 7. Thus, substantial increases in miR-720 levels can be detected early, prior to the increase in tumor growth when tumor sizes are fairly small.

The MP domain alone or the entire ectodomain of ADAM8 has been shown to be secreted from cells expressing ADAM8 [22]. This soluble fraction of ADAM8 was recently found to be shed from malignant breast tumors into the blood of patients [24]. Given that miR-720 levels were significantly higher in the blood of mice bearing ADAM8-positive TNBC tumors, we next asked whether miR-720 levels are elevated in the serum of TNBC patients with detectable ADAM8 in the blood versus normal individuals. Serum samples were obtained from consenting TNBC patients taken at the time of diagnosis, before any treatment was provided, and from normal healthy individuals. Samples were first analyzed for levels of soluble ADAM8 protein shed into the blood using an ELISA kit (R&D Systems). ADAM8 levels were significantly higher in serum samples of TNBC patients in comparison to normal healthy individuals (Fig. 9a). Interestingly, 78 % (29/37) of the TNBC patients had ADAM8 levels above the median value observed in healthy individuals, whereas levels of eight TNBC patient serum samples were below the medium value (Fig. 9b). Next, RNA was extracted from the serum samples using a Macherey-Nagel Nucleospin miRNA plasma kit and subjected to RT-PCR for miR-720. Unfortunately, RNA yield and quality from three of the samples were poor as judged by RNA integrity analysis using an Agilent bioanalyzer, and these samples had to be excluded from the analysis. hsa-miR-16 was used to normalize the data as its levels remained unchanged in normal versus patient samples (Additional file 3: Figure S2), similar to previous reports by others in healthy individuals versus breast cancer patients [58]. As seen in Fig. 9c, levels of miR-720 were significantly higher in patient samples with elevated amounts of ADAM8, whereas normal healthy individuals and TNBC patients with comparably low ADAM8 levels displayed low levels of miR-720. This increase was not correlated with tumor size (Additional file 3: Figure S2). Thus, higher levels of miR-720 are found in the serum of TNBC patients with high ADAM8 serum levels.