Intratumoral Heterogeneity of ADAM23 Promotes Tumor Growth and Metastasis Through LGI4 and Nitric Oxide Signals

ORIGINAL ARTICLE Intratumoral heterogeneity of ADAM23 promotes tumor growth and metastasis through LGI4 and nitric oxide signals

ET Costa1,2, GF Barnabé1,2,MLi3, AAM Dias4, TR Machado2, PF Asprino1,2, FP Cavalher2, EN Ferreira5, M del Mar Inda3, MH Nagai6, B Malnic6, ML Duarte1,2, KRM Leite7, ACSD de Barros8, DM Carraro5, R Chammas7, HA Armelin6,9, W Cavenee3, F Furnari3 and AA Camargo1,2

Intratumoral heterogeneity (ITH) represents an obstacle for cancer diagnosis and treatment, but little is known about its functional role in cancer progression. The A Desintegrin And Metalloproteinase 23 (ADAM23) gene is epigenetically silenced in different types of tumors, and silencing is often associated with advanced disease and metastasis. Here, we show that invasive breast tumors exhibit significant ADAM23-ITH and that this heterogeneity is critical for tumor growth and metastasis. We demonstrate that while loss of ADAM23 expression enhances invasion, it causes a severe proliferative deficiency and is not itself sufficient to trigger metastasis. Rather, we observed that, in ADAM23-heterotypic environments, ADAM23-negative cells promote tumor growth and metastasis by enhancing the proliferation and invasion of adjacent A23-positive cells through the production of LGI4 (Leucine-rich Glioma Inactivated 4) and nitric oxide (NO). Ablation of LGI4 and NO in A23-negative cells significantly attenuates A23-positive cell proliferation and invasion. Our work denotes a driving role of ADAM23-ITH during disease progression, shifting the malignant phenotype from the cellular to the tissue level. Our findings also provide insights for therapeutic intervention, enforcing the need to ascertain ITH to improve cancer diagnosis and therapy.

Oncogene advance online publication, 24 March 2014; doi:10.1038/onc.2014.70

INTRODUCTION lung32 tumors. We have previously demonstrated that ADAM23 Cancer is a dynamic heterogeneous disease. The coexistence of expression is silenced by DNA promoter hypermethylation in the 26,27,30 tumor cells with different phenotypic traits within a primary tumor advanced stages of breast and head and neck tumors. In has been known since the late 1970s. This phenotypic diversity, breast cancer, we showed that ADAM23 hypermethylation is an called intratumoral heterogeneity (ITH), appears to be the end independent prognostic factor associated with a ninefold higher 27 result of branched evolutionary tumor growth, fostered by risk of developing distant metastases and lower survival rates. In increased genetic and epigenetic instability and selective pres- addition, we demonstrated that knockdown of ADAM23 expression sures from the tumor environment.1–8 in the MDA-MB-435 cell line enhanced tumor cell migration, 27 Recent studies, mostly using deep-sequencing technologies, adhesion and arrest in the lungs of immunodeficient mice. have elucidated the genetic basis of ITH in different types of Here, we show for the first time that loss of ADAM23 expression, tumors and associated metastatic sites.9–19 Collectively, these previously associated with the development of distant metastases studies have demonstrated that ITH is a ubiquitous characteristic and poor prognosis, causes a proliferative and colonization of neoplasms, arising during disease progression, and that primary deficiency and, counterintuitively, is not sufficient to trigger tumors are ecosystems of evolving clones with different spatial metastatic dissemination. Rather, the rise of ADAM23-negative neg and temporal distributions.20–22 (A23 ) cells within an ADAM23-positive tumor generates Although ITH has been an underlying concept in cancer biology ADAM23-ITH and actively enhances proliferation and promotes for decades,1–8,23 it is not yet fully considered by clinicians and invasion of adjacent ADAM23-positive (A23pos) tumor cells through pathologists and represents a major obstacle for cancer diagnosis Leucine-rich Glioma Inactivated 4 (LGI4) secretion and nitric oxide and treatment. This is due mainly to technical limitations in (NO) production, respectively. Ablation of LGI4 and NO production assessing ITH in the clinical setting and to our rudimentary uncouples this cellular crosstalk and attenuates tumor cell knowledge of the functional role of ITH for cancer progression, proliferation and invasion. Our findings highlight a driving drug resistance and metastasis. role of ADAM23-ITH in cancer progression by promoting tumor The ADAM23 gene (also known as MDC3), encoding a growth and metastasis. Our results also provide important insights non-catalytically active member of A Disintegrin And for therapeutic intervention and enforce the need to address Metalloproteinase family,24 is frequently silenced in gliomas,25 tumor subclonal architecture to improve the diagnosis and breast,26,27 pancreatic,28 gastric,29 head and neck,30 colorectal31 and treatment of cancer.

1Centro de Oncologia Molecular, Hospital Sírio Libanês, São Paulo, Brazil; 2Ludwig Institute for Cancer Research (LICR), São Paulo, Brazil; 3Ludwig Institute for Cancer Research (LICR), University of California, San Diego, CA, USA; 4Departamento de Biologia Geral (ICB), Universidade Federal de Minas Gerais, Belo Horizonte, Brazil; 5Centro Internacional de Pesquisa, Hospital AC Camargo, São Paulo, Brazil; 6Departamento de Bioquímica (IQ), Universidade de São Paulo, São Paulo, Brazil; 7Departamento de Urologia, Faculdade de Medicina, Universidade de São Paulo, São Paulo, Brazil; 8Departamento de Mastologia, Hospital Sírio Libanês, São Paulo, Brazil and 9Instituto Butantan, São Paulo, Brazil. Correspondence: Dr AA Camargo, Ludwig Institute for Cancer Research (LICR), Molecular Oncology Center, Hospital Sírio Libanês, Rua Cel. Nicolau dos Santos 69, São Paulo 01308-060, Brazil. E-mail: [email protected] or [email protected] Received 12 July 2013; revised 6 January 2014; accepted 14 January 2014 ADAM23-ITH promotes tumor growth and metastasis ET Costa et al 2 RESULTS These results indicate that ADAM23 expression is down- ADAM23 silencing and ITH in invasive breast carcinomas regulated during breast cancer progression and that ADAM23- To explore the role of ADAM23 silencing during breast ITH is a common characteristic of invasive carcinomas. cancer progression, we first analyzed if there were differences in ADAM23 mRNA expression levels between ductal breast carcinomas in situ (DCIS) and invasive ductal breast carcinomas ADAM23-ITH promotes tumor cell proliferation and tumor growth (IDCs). Compared with normal breast, ADAM23 levels were To address the functional relevance of ADAM23-ITH during cancer reduced in most IDC (10/19), but not in DCIS (1/7) (Figure 1a, progression, we first examined the role of ADAM23-ITH in tumor Po0.05). We also analyzed the ADAM23 mRNA expression cell proliferation using three-dimensional cultures, which more levels in paired in situ and invasive components of four effectively mimic tumor behavior,33 as well as by subcuta- IDCs to determine if ADAM23 silencing is homogeneous neous tumorigenesis assays. For these experiments, we used the within IDC. Tumor cells from both components were laser MDA-MB-435 cell line, which we have previously used to address microdissected (Figure 1b) and the heterogeneous ADAM23 the functional impact of ADAM23 silencing in tumor cell adhesion expression was observed in two out of the four tumor samples and migration.27 analyzed (IDC2 and IDC4), most pronounced in tumor IDC4, In vitro, MDA-MB-435 ADAM23-positive cells (435-A23pos)or extracted from a patient with extensive lymph node metastasis their ADAM23 short hairpin RNA (shRNA) derivatives (435-A23neg) (Figure 1c). (Supplementary Figures 2a and b) and a mixture of 1:1 ratio of ADAM23 protein expression was also analyzed in 12 indepen- both (435-A23het) were cultured as multicellular spheroids (MS), dent IDCs. As illustrated in Figure 1d, ADAM23 protein staining and proliferation rates were estimated by bromodeoxyuridine was more prominently detected in breast ducts with preserved (BrdU) labeling. A lower percentage of BrdU-labeled cells was normal architecture and a gradual decrease in staining intensity observed in 435-A23neg-MS (21%) compared with 435-A23pos-MS was observed between the in situ and invasive components of (32%) and 435-A23het-MS (32%, Po0.01) (Figure 2a). In agreement different IDCs (Figure 1d). It is worth noting that ADAM23-ITH with the proliferative deficiency of 435-A23neg cells, using was observed in topographically distinct invasive areas within anchorage-independent clonogenic assays, we observed that undifferentiated IDCs, with some invasive areas being composed 435-A23neg cells formed an equivalent number of colonies to by a mosaic cluster of A23pos and A23neg tumor cells (Figure 1e, in 435-A23pos cells, although these colonies were on average detail, and Supplementary Figure 1). threefold smaller, confirming that both cells have a similar

Figure 1. ADAM23 expression levels in ductal breast carcinomas. (a)RT–qPCR analysis of ADAM23 mRNA expression in normal breast (NB) (n = 4), DCIS (n = 7) and IDCs (n = 19). (b) Representative example of a laser capture microdissection of the paired in situ (top panels) and invasive (bottom panels) components of an IDC. (c)RT–qPCR analysis of ADAM23 mRNA expression in paired in situ and invasive components of four IDCs (IDC1–4). Expression levels relative to normal breast tissue and normalized to the hypoxanthine guanine phosphoribosyltransferase (HPRT) endogenous control gene are shown in (a and c). (d and e) Immunohistochemistry staining of ADAM23 protein was carried out using the polyclonal antibody anti-ADAM23 (HPA012130). (d) ADAM23 protein expression in mammary normal ducts, in situ and invasive tumor components of an IDC (IDC15 from patient no. 15). Bar represents 100 μm. (e) ADAM23-ITH is observed between topographically distinct invasive areas of an undifferentiated IDC. In detail, mosaic clusters of A23pos (green arrow) and A23neg (red arrow) tumor cells coexisting in close proximity within an invasive component. Bar represents 50μm.

Figure 2. ADAM23-ITH promotes cell proliferation and tumor growth. (a) 435-A23pos, 435-A23neg or a 1:1 mixture of both (435-A23het) were cultured as MS for 4 days and were pulsed for 6 h with BrdU. After MS dissociation, cell proliferation was evaluated by flow cytometric analysis of BrdU labeling (n = 10 000 cells per MS, 8 MS per group). NS, nonsignificant, **Po0.01 using one-way analysis of variance (ANOVA). (b and c) Cells were plated at clonogenic density in 0.6% soft agar and left for 2 weeks to allow colony formation. (b) The number of colonies formed per well (n = 3 independent experiments) and (c) the average colony size (area, mm2)(n = 20 colonies per experiment) were determined under the light microscope. ***Po0.001 using Student’s t-test. (d and e) 435-A23pos, 435-A23neg or 435-A23het mixtures were inoculated subcutaneously (s.c.) in female nude mice. (d) Kaplan–Meier curves depict s.c. tumor-free mice. Arrow indicates reduced tumor latency of 435-A23het tumors. ***Po0.001 using a Mantel–Cox log-rank test. (e) S.c. tumor growth was monitored weekly by measuring two perpendicular tumor diameters. Arrow indicates the early increase in growth of 435-A23het tumors. ***Po0.001 using one-way ANOVA. pos pos neg (f and g) Red fluorescent 435-A23 cells (435-A23 /DiIC18) were mixed at different ratios with unlabeled 435-A23 , allowed to aggregate pos het as MS and proliferation was evaluated as described above. (f) Proliferation rates of 435-A23 /DiIC18 cells in 435-A23 -MS (n = 10 000 DiIC18- labeled cells per MS, 6 MS per group). (g) Proliferation rates of 435-A23neg cells in 435-A23het-MS (n = 10 000 unlabeled cells per MS, 6 MS per group). NS, nonsignificant, *Po0.05 and ***Po0.001 using one-way ANOVA. Error bars indicate s.e.m. in all experiments. clonogenic potential but distinct proliferative capacity in vitro expression in 435-A23het tumors (Supplementary Figure 4). These (Figures 2b and c, Po0.001). Accordingly, ADAM23 overexpres- results indicate that the coexistence of A23neg and A23pos cells in sion in CMS5a cells significantly promoted colony growth primary tumors enhance proliferation and tumor growth. in anchorage-independent clonogenic assays (Supplementary To explore this possibility, we produced 435-A23het-MS Figure 3). In subcutaneous tumorigenesis assays, 435-A23neg cells composed of 435-A23pos cells prelabeled with a red fluorescent pos showed impaired tumorigenesis (5/15, 33%) when compared with dye (435-A23 /DiIC18) and different ratios of unlabeled 435- 435-A23pos (14/15, 93%, Po0.01) and 435-A23het (10/11, 91%, A23neg cells, so that we could track the proliferation rate of both Po0.01) cells (Figure 2d). Significant differences in tumor growth cell subpopulations in heterotypic MS. In 435-A23het-MS, the pos kinetics and final tumor sizes were also observed between 435- percentage of BrdU-labeled 435-A23 /DiIC18 cells increased from A23neg and the other groups (Figure 2e, Po0.001). Interestingly, 28 to 45% along with the increase in the number of 435-A23neg histological analysis of 435-A23neg injection sites from tumor-free cells (Figure 2f, Po0.001), indicating that, in heterotypic environ- mice revealed the presence of a small group of living tumor cells ments, 435-A23neg cells enhance the proliferation rate of 435- for periods up to 10 weeks after injection. These ADAM23- A23pos cells. In contrast, rates of BrdU labeling in 435-A23neg cells negative ‘dormant’ tumors exhibited a late and slow growth progressively decreased from 20 to 10%, with the gradual increase pos 12 weeks after inoculation, and the real-time polymerase chain in 435-A23 /DiIC18 cell number (Figure 2g, Po0.05). reaction (PCR) analysis revealed that ADAM23 silencing was stably To demonstrate that ADAM23-dependent proliferative signals maintained in these tumors during late in vivo growth (data not are not restricted to MDA-MB-435 cancer cells, we screened a shown). panel of tumor cell lines for ADAM23 mRNA expression It is worth noting that animals injected with 435-A23het mixtures (Supplementary Figure 5) and performed similar experiments exhibited reduced tumor latency than animals injected with either with SK-Mel-37 melanoma cells (SK-A23pos) and their ADAM23- 435-A23pos or 435-A23neg cells (Figure 2d) and, in vitro, 435-A23het- shRNA derivatives (SK-A23neg) (Supplementary Figures 2c and d). MS had similar percentages of BrdU-labeled cells as 435-A23pos- In vitro, SK-A23neg-MS showed a smaller percentage of BrdU- MS, despite having initially half the amount of 435-A23pos cells labeled cells than SK-A23pos-MS (Supplementary Figure 6a). In vivo, (Figure 2a). Flow cytometry analysis of tumors from mice injected subcutaneous tumorigenicity of SK-A23neg cells (5/7) was not with 435-A23het mixtures revealed that 435-A23neg cells were impaired compared with SK-A23pos cells (5/7), although significant retained in these tumors 10 weeks after inoculation, despite differences in growth kinetics and tumor size were observed their proliferation deficiency (data not shown). Similar results (Supplementary Figure 6b, Po0.01), Notably, SK-A23het mixtures were observed by immunohistochemical analysis of ADAM23 were more tumorigenic (8/8) and showed reduced tumor latency

© 2014 Macmillan Publishers Limited Oncogene (2014), 1 – 10 ADAM23-ITH promotes tumor growth and metastasis ET Costa et al 4 compared with both homotypic groups (Supplementary Figure 6c, cells with or without ablation of LGI4 (435-A23neg/shLGI4 or 435- Po0.05). A23neg/shCtrl, respectively) (Figure 3b). Altogether, our findings demonstrate that ADAM23 expression Consistent with a proproliferative function of LGI4, the is important for tumor growth at the primary site and that percentage of BrdU labeling in 435-A23neg/shLGI4-MS (20%) was ADAM23 silencing is associated with an accentuated deficiency in significantly lower than in 435-A23neg/shCtrl-MS cells (28%) and cell proliferation, but not with reduced clonogenic cell survival. 435-A23pos-MS (34%) (Figure 3c, Po0.001). Moreover, the pos Moreover, we demonstrated that in ADAM23-heterotypic environ- 435-A23 /DiIC18 subpopulation showed an increased percentage ments, the proliferation of the A23pos subpopulation is enhanced of BrdU labeling, from 32 to 40%, when cocultured with by A23neg cells, compensating for the intrinsic proliferative 435-A23neg/shCtrl cells (Po0.001), but not when cocultured with deficiency of the latter subpopulation. Most importantly, our 435-A23neg/shLGI4 cells (31%, Figure 3d). As expected, LGI4 knock- results suggest that ADAM23-ITH can promote tumor growth. down in 435-A23neg cells significantly impaired by 43% the previously observed enhanced growth of A23het subcutaneous tumors (Figure 3e). In agreement with an ADAM23-mediated LGI4 mediates the proliferative crosstalk in ADAM23- proliferative crosstalk, LGI4-dependent tumor growth was heterotypicenvironments exclusively determined by the coexistence of A23neg and LGI4 (also known as LGIL3), a member of the LGI family, is a putative A23pos cells, as subcutaneous inoculums of 435-A23neg/shCtrl or secreted protein34 involved in proliferation in the nervous system35 435-A23neg/shLGI4 alone were essentially non-tumorigenic, because and a known ADAM11, ADAM22 and ADAM23 ligand.35–37 We of the absence of ADAM23 expression. found that LGI4 mRNA expression was eightfold higher in 435- To our knowledge, this is the first demonstration of the A23neg than in 435-A23pos cells (Figure 3a, Po0.01). To determine relevance of LGI4/ADAM23 interactions for tumor cell proliferation whether LGI4 produced by 435-A23neg cells is responsible for the and tumorigenesis. Moreover, our results show that LGI4 is the key enhanced cellular proliferation of 435-A23pos cells in 435-A23het-MS, factor mediating the proliferative crosstalk between A23neg and we examined BrdU labeling in A23het-MS composed of 435-A23neg A23pos cells in ADAM23-heterotypic environments.

Figure 3. LGI4 mediates the proliferative crosstalk in ADAM23-heterotypic environments. (a)RT–qPCR analysis of LGI4 expression in 435- A23pos and 435-A23neg cells. Expression levels relative to MDA-MB-435 wild-type cells and normalized to hydroxymethylbilane synthase (HMBS) endogenous control gene are shown (n = 4). **Po0.01 using Student’s t-test. (b) 435-A23neg cells were stably transduced with two different LGI4 shRNAs sequences (shLGI4 nos. 1 or 2) or with control shRNA (shCtrl) and LGI4 mRNA expression levels relative to cells transfected with shCtrl were determined as described above (n = 3). ***Po0.001 using one-way analysis of variance (ANOVA). (c) 435-A23pos and 435-A23neg cells transfected with shLGI4 (A23neg/shLGI4) or shCtrl (A23neg/shCtrl) were allowed to aggregate as MS and proliferation was evaluated as described above (n = 10 000 cells per MS, 6 MS per group). ***Po0.001 using one-way ANOVA. (d) Proliferation of 435-A23pos/ het neg/shCtrl DiIC18 cells in 435-A23 -MS composed of 90% of A23 or A23neg/shLGI4 cells was determined as described above (n = 10 000 cells per MS, 4 MS per group). NS, nonsigniﬁcant, ***Po0.001 using one-way ANOVA. (e) Subcutaneous A23het tumor growth constituted of 8 × 105 435-A23neg cells stably transduced with two LGI4 shRNAs sequences (grouped as shLGI4) or with shCtrl and 2 × 105 435-A23pos cells. A23- homotypic inoculums (8 × 105 435-A23neg or 2 × 105 435-A23pos cells) were included as controls. **Po0.01 using one-way ANOVA. Error bars indicate s.e.m. in all experiments.

Oncogene (2014), 1 – 10 © 2014 Macmillan Publishers Limited ADAM23-ITH promotes tumor growth and metastasis ET Costa et al 5 ADAM23-ITH promotes tumor cell invasion and metastasis into the mammary fat pad of immunodeficient mice. As expected, We next examined the role of ADAM23-ITH in tumor cell invasion the percentage of animals that developed tumors was lower neg and metastasis using MS embedded in collagen matrices, as well in mice inoculated with 435-A23 cells (9/12, 75%) than with pos as spontaneous metastasis assays. 435-A23 cells (14/14, 100%, Po0.05, Figure 4c), but no Ten days after embedment in collagen matrices, 435-A23neg-MS significant differences in tumor growth, final sizes or tumor were visibly more invasive than 435-A23pos-MS (Figure 4a). The latency were observed between the groups when inoculated average speed of invasion in 435-A23neg-MS was up to four times with the Matrigel into the mammary fat pad (Figures 4c and d). higher than in 435-A23pos-MS (4.7 and 1.6 μm/h, respectively, Unexpectedly, as ADAM23 silencing has been previously asso- 27 Po0.01, Figure 4b). It is worth noting that an invasive phenotype ciated with metastasis development and poor prognosis, was accomplished in the noninvasive SK-Mel-37 cell line after we observed no significant differences in the number of animals neg ADAM23 ablation. The average invasion speed was 4.5 times that developed metastases between the 435-A23 (2/12, 16%) pos higher (Po0.01) for SK-A23neg-MS than for SK-A23pos-MS and 435-A23 (5/14, 36%, P>0.05) groups (Table 1). As observed (Supplementary Figure 7). Accordingly, ADAM23 overexpression for the subcutaneous tumorigenesis assays, ADAM23 silencing in CMS5a cells significantly inhibited invasion in collagen matrices remained stable throughout the spontaneous metastasis assay (Supplementary Figure 8). and, interestingly, metastatic lesions from animals bearing het We also carried out spontaneous metastasis assays by inoculat- 435-A23 tumors displayed either a positive or negative pattern ing 435-A23pos, 435-A23neg and a 1:1 mixture of both (435-A23het) of ADAM23 mRNA expression, probably associated with the

Figure 4. ADAM23-ITH promotes cell invasion and metastasis. (a) Representative images of 435-A23pos-MS, 435-A23neg-MS and 435-A23het-MS embedded in collagen matrices for 10 days. (b) Invasion speed of MS presented in (a)(n = 4 MS per group). **Po0.01, ***Po0.001 using one- way analysis of variance (ANOVA). (c and d) 435-A23pos, 435-A23neg or a 1:1 mixtures (435-A23het) were inoculated into the mammary fat pad (m.f.p.) of female nude mice. (c) Kaplan–Meier curves depict m.f.p. tumor-free mice. *Po0.05 using a Mantel–Cox log-rank test. (d) Tumor growth in the m.f.p. by measuring two perpendicular tumor diameters. (e) Invasion speed of 435-A23het-MS as a function of the percentage of 435-A23neg cells in the MS. *Po0.05, ***Po0.001 using one-way ANOVA. (f) Representative images of the invasiveness of 435-A23pos/GFP cells in 435-A23pos-MS (top panel) or in 435-A23het-MS (bottom panel). Red arrows highlight the distances traveled by 435-A23pos/GFP cells. The graph below represents the invasion speed of 435-A23pos/GFP cells in both environments (n = 4 MS per group). **Po0.01 using Student’s t-test. (g) Invasive bundles were formed by 435-A23pos/GFP and 435-A23neg/RFP. Invasive cells were outlined and reconstructed to compose a merged picture (detail). (h) Total number of migrating cells when 435-A23pos or 435-A23neg cells or 435-A23het mixtures were plated in the upper chamber of transwell (n = 4). ***Po0.001 using one-way ANOVA. (i) Number of 435-A23pos/GFP migrating cells when cocultured in the presence of either 435-A23pos or 435-A23neg cells (n = 3). *Po0.05 using Student’s t-test. (j) Number of 435-A23pos/GFP migration cells toward serum-free 24h conditioned medium (24 h-CM) from 435-A23pos or 435-A23neg cells (n = 3). ***Po0.001 using one-way ANOVA. Error bars indicate s.e.m. in all experiments. NS, nonsigniﬁcant.

© 2014 Macmillan Publishers Limited Oncogene (2014), 1 – 10 ADAM23-ITH promotes tumor growth and metastasis ET Costa et al 6 Altogether, our findings demonstrate that A23neg cells are Table 1. Metastatic potential of 435-A23pos, 435-A23neg and highly invasive, but not sufficient to seed metastasis and that, in 435-A23het tumors an ADAM23-heterotypic environment, the invasive and migratory pos neg Mice with lung metastasis behavior of A23 cells is enhanced by close proximity to A23 cells. Most importantly, our results suggest that ADAM23-ITH 435-A23neg 2/12 (16%) positively affects tumor cell motility, invasion and, consequently, 435-A23pos 5/14 (36%)NS metastasis. 435-A23het 11/23 (48%)* Abbreviation: NS, nonsignificant. 435-A23pos or 435-A23neg cells or a 1:1 NO mediates the invasive crosstalk in ADAM23-heterotypic het mixture of both (435-A23 ) were suspended in the Matrigel and injected environments into the mammary fat pad of female Balb/c nude mice (106 cells in 0.05 ml NO is a transient species that exerts its physiological effects at of the Matrigel). Primary tumors were surgically removed and, after 38 8 weeks, animals were killed and the number of metastasis in lungs, short distances (up to 50 cell diameters). Considering the close kidneys and bladders were assessed by macroscopic observation at × 10 proximity dependence of ADAM23-ITH for the invasive crosstalk magnification. *P ⩽ 0.05 using a χ2 test. and the important role of NO in tumor metastasis by promoting cell migration and invasion,39–41 we analyzed the expression of NO synthases (NOS1, NOS2 and NOS3) in A23pos and A23neg cells. ’ We found that NOS3 expression (also known as eNOS) was different ADAM23 expression status of their founder s tumor cells neg pos (Supplementary Figure 9). upregulated in 435-A23 cells compared with 435-A23 cells o It is worth noting that the metastatic potential of 435-A23het (Supplementary Figure 10, P 0.01), increasing NO levels by 56% (Figures 5a and b, Po0.01). Similarly, SK-A23neg cells produced tumors was threefold higher than that of tumors composed pos exclusively of 435-A23neg cells (11/23, 48%, P ⩽ 0.05) (Table 1) and, threefold more NO than SK-A23 cells (Supplementary Figure 11, het Po0.001). To examine the effect of exogenous NO on the in vitro, 435-A23 -MS had a similar invasive behavior to 435- pos A23neg-MS, despite having initially half the amount of 435-A23neg migratory phenotype of A23 cells, we performed haptotactic cells (Figure 4b, Po0.001). In addition, we observed a positive assays in the presence of the slow-releasing NO donor SNAP (S- neg nitroso-N-acetylpenicillamine). A 25% significant increase in the correlation between the percentage of A23 cells within the MS pos and the average invasion speed (Figure 4e, r = 0.67), indicating number of 435-A23 migrating cells was observed at low SNAP o that ADAM23-ITH can enhance tumor invasion and metastasis. concentrations (Figure 5c, P 0.01). In agreement with this, To explore this possibility, we produced A23het-MS, composed pharmacological inhibition of NOS with L-NAME (L-NG-nitroargi- fi of GFP-labeled 435-A23pos cells (435-A23pos/GFP) and RFP-labeled nine methyl ester) signi cantly inhibited the migration of 435- pos neg fi 435-A23neg cells (435-A23neg/RFP), so that we could track the A23 cells in the presence of 435-A23 cells, con rming that distance traveled daily over 12 days by both cell subpopulations in NO is a key mediator of ADAM23-mediated cell migration (Supplementary Figure 12). the heterotypic spheroids. We observed an increase in 60% in the neg pos/GFP het To investigate the effect of NO secreted by A23 cells on the invasion speed of 435-A23 cells in A23 -MS compared pos pos invasive behavior of A23 cells, we ablated NOS3 expression with that in A23 -MS (composed of labeled and unlabeled 435- neg neg/shNOS3 A23pos cells) (2.5 and 1.5 μm/h, respectively; Figure 4f, Po0.01). in 435-A23 cells using NOS3 shRNAs (435-A23 ; No difference in the invasion speed of 435-A23neg/RFP cells within Figure 5d). Whereas the 30% reduction in endogenous NO o A23het-MS or A23neg-MS was observed (data not shown). Consistent production (Figure 5e, P 0.01) did not alter the intrinsic invasive behavior of 435-A23neg cells (data not shown), it was sufficient to with a cooperative invasive pattern, we frequently noticed neg pos/GFP neg/RFP ablate completely the influence of 435-A23 cells on the invasive interlaced subpopulations of 435-A23 and 435-A23 pos het cells within single-cell bundles from A23het-MS (Figure 4g). behavior of 435-A23 cells in A23 -MS (Figures 5f and g, o pos/GFP Next, we performed haptotactic migration assays to determine P 0.01). Consistently, the transwell migration of 435-A23 pos cells increased by 60% when they were cocultured with 435- whether the increased invasive behavior of A23 cells is strictly neg/shCtrl 2 neg/shNOS3 dependent on the generation of a less resistant migratory A23 (39 cells per mm ) compared with 435-A23 2 o pathway by A23neg cells in collagen matrices, or if A23neg cells cells (24 cells per mm ; Figure 5h, P 0.01). also actively influence the migratory behavior of A23pos cells These results demonstrate that NO production is upregulated in A23neg cells and NO is a key messenger mediating the invasive through paracrine signals. We observed that the number of pos neg migrating cells was higher in 435-A23het (250 cells per mm2) than crosstalk between 435-A23 and 435-A23 cells in 435-A23pos cultures (22 cells per mm2, Po0.001), but no significant differences were observed with 435-A23neg (205 cells per mm2, P > 0.05; Figure 4h). Considering that A23het DISCUSSION mixtures contained half the number of A23neg cells, these results Here we showed, for the first time, that ADAM23 silencing can confirm that when mixed A23pos and A23neg cells have a different elicit a phenotypic switch from a proliferative to an invasive migratory behavior. phenotype in tumor cells. Whereas A23pos tumor cells exhibit a We then carried out haptotactic assays to analyze the migratory fast-proliferating phenotype, high tumorigenicity and a lower behavior of 435-A23pos/GFP cells when mixed with unlabeled 435- invasive potential, A23neg tumor cells display a highly invasive A23pos or 435-A23neg cells in the upper chamber of transwell phenotype, but an increased susceptibility to the trophic effect of plates. Notably, a 77% increase in the number of 435-A23pos/GFP the environment for optimal tumor growth. We also demon- migrating cells was observed when they were mixed with 435- strated, at both the mRNA and protein level, that ADAM23 A23neg cells (from 40 to 77 cells per mm2; Figure 4i, Po0.05). expression is downregulated during breast cancer progression Finally, we compared the chemotactic activity of 24 h conditioned and ADAM23-ITH is a common characteristic of invasive breast medium obtained from 435-A23pos and 435-A23neg cells. We carcinomas. ADAM23-ITH was observed in topographically distinct observed no differences in the chemotactic response of 435-A23pos invasive areas of undifferentiated IDCs, with invasive components cells to both conditioned medium, but a threefold increase in the being frequently composed by mosaic clusters of A23pos tumor number of 435-A23pos migrating cells with both conditioned cells coexisting in close proximity with A23neg cells. medium compared with control medium (Figure 4j, Po0.001), Surprisingly, however, we observed that A23neg cells are not suggesting that the enhancing effect of 435-A23neg cells on the sufficient to seed metastasis owing to a severe deficiency migratory behavior of 435-A23pos cells depends on close proximity. to proliferate. Instead, the rise of A23neg cells within an

Figure 5. NO mediates cooperative invasion in ADAM23-heterotypic environments. Quantification of NO production was performed using 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM) as described in Materials and methods. (a) Representative histograms of the quantification of NO production in 435-A23pos and 435-A23neg cells. The gray histogram represents the background fluorescence of cells without DAF-FM. (b) NO production of 435-A23pos and 435-A23neg cells (n = 9 per group). **Po0.01 using Student’s t-test. (c) Migration assays were carried out in the presence of the increasing concentrations of the slow-releasing NO donor S-nitroso-N-acetylpenicillamine (SNAP) (n = 2 per SNAP concentration). **Po0.01 using one-way analysis of variance (ANOVA). (d) 435-A23neg cells were stably transduced with two different NOS3 shRNAs sequences (shNOS3 nos. 1 or 2) or with control shRNA (shCtrl) and NOS3 mRNA expression levels relative to cells transfected with shCtrl and normalized to hydroxymethylbilane synthase (HMBS) endogenous control gene were determined by RT–qPCR (n = 3). *Po0.05 using one-way ANOVA. (e) NO production in 435-A23neg/shNOS3 or 435-A23neg/shCtrl cells was measured as described above (n = 4 per group). **Po0.01 using Student’s t-test. (f) Representatives images of the invasive behavior of 435-A23pos/GFP cells in 435-A23pos-MS or in 435-A23het-MS composed of either 435-A23neg/shCtrl or 435-A23neg/shNOS3 cells. Red arrows highlight the distances traveled by 435-A23pos/ GFP cells in different environments. (g) Invasion speed of 435-A23pos/GFP cells in 435-A23pos-MS, 435-A23neg/shCtrl-MS or 435-A23neg/shNOS3-MS (n = 6 MS per group). **Po0.01 and ***Po0.001 using one-way ANOVA. (h) Number of 435-A23pos/GFP migrating cells when cocultured in the presence of either 435-A23neg/shCtrl or 435-A23neg/shNOS3 unlabeled cells (n = 5). **Po0.01 using Student’s t-test. Error bars indicate s.e.m. in all experiments. NS, nonsignificant.

ADAM23-positive tumor generates ADAM23-ITH that drives tumor The potential of NOS3 as a therapeutic target has recently been growth and metastasis by actively enhancing the proliferative and reconsidered44 and could also be particularly useful in the context invasive behavior of adjacent highly tumorigenic A23pos cells. of an ADAM23-heterotypic environment. Unusual NO production Moreover, we demonstrated that LGI4 and NO are the key factors has been detected in different types of tumors, altering the cell produced by A23neg tumor cells enhancing proliferation and redox state and signaling. Tumor-derived NO has been shown to invasion, respectively, of adjacent A23pos cells in heterotypic tumors. enhance migration and invasion of cancer cells.39–41 Notably, It has been proposed that ITH may drive tumor progression by NOS3-ITH has been observed in primary breast tumors, and higher creating an ecological niche in which tumor cells with different levels of NOS3 were detected in undifferentiated tumor zones genotypes interact through paracrine signals and cooperate to than in differentiated areas.40 promote tumor growth and dissemination.7 Identifying the key In conclusion, based on our previous findings26,27 and on the molecules mediating the cellular cooperation is thus promising work described here, we suggest that ADAM23 silencing, avenues to reveal new approaches for the effective treatment of observed during breast cancer progression as a result of tumor metastatic disease. Interactions between LGI and ADAM proteins epigenetic instability, generates a subpopulation of ADAM23- are known to promote proliferation and differentiation of negative clones within ADAM23-positive tumors and that meta- neuronal precursors cells and adipocytes.42,43 However, the static spread and malignancy is in fact driven by the coexistence findings reported here indicate that LGI/ADAM interactions are and interaction between ADAM23-negative and -positive sub- also relevant for tumorigenesis and ablation of LGI4 production in populations in the primary tumor. In this scenario, it is tempting to A23neg cells significantly attenuates proliferation and tumor speculate that the previously observed association between growth in the A23-heterotypic environments. ADAM23 hypermethylation and poor clinical outcome27 is indeed Curiously, changing the ratio of A23pos and A23neg cell associated with ADAM23-ITH and to propose that additional subpopulations in A23het-MS significantly altered the proliferative studies addressing the association between ADAM23-ITH in differences between them. One explanation for these observa- primary tumors, metastatic disease and clinical outcome are tions is that both subpopulations are competing for LGI4- worth pursuing. dependent proliferative signals, and ADAM23 expression confers an inherent advantage in this competitive environment. Moreover, considering that MDA-MB-435 cells do not express ADAM11 MATERIALS AND METHODS mRNA, but do express ADAM22 mRNA (Supplementary Figure 13), Laser microdissection we speculate that the residual LGI4-dependent proliferation of Tumor cells were laser captured using the Pix Cell-II LCM System (Arcturus- 435-A23neg cells is supported by LGI4/ADAM22 interactions. Engineering Inc., Mountain View, CA, USA) as described previously.45

© 2014 Macmillan Publishers Limited Oncogene (2014), 1 – 10 ADAM23-ITH promotes tumor growth and metastasis ET Costa et al 8 Samples were free of stromal cells contamination. Breast DCIS samples are Sodium dodecyl sulfate–polyacrylamide gel electrophoresis/ composed of tumor cells captured from ducts of pure DCIS lesion. western blot IDC samples were divided geographically into two components: in situ and Quantified membrane extracts were resolved on a 10% sodium dodecyl invasive components. In situ components are composed of tumor cells sulfate–polyacrylamide gel electrophoresis gel, transferred to poly- captured from mammary ducts and invasive components are composed of vinylidene fluoride membrane (GE Healthcare Biosciences, Pittsburgh, fi in ltrative tumor cells captured outside from the ducts. These experiments PA, USA) and probed with antibody to ADAM23 (HPA012130; were conducted according to ethic standards and approved by the Ethics Sigma-Aldrich Co.) or to αv-integrin (AB1930; Millipore Co., Billerica, MA, Committee in Human Research from FAP-AC Camargo (1143/08). USA) antibodies. Membranes were incubated for 60 min at room temperature in 0.1% Tween-20 with the secondary HRPT-conjugated antibody. Reactive proteins were visualized with the ECL Western Reverse transcription and quantitative PCR Blotting Detection System (GE Healthcare Bioscience) according to the Total RNA was extracted using Trizol (Life Technologies, New York, NY, manufacturer's recommendations. USA). Reverse transcription (RT) was carried out with 2 μg DNA-free total RNA using Superscript II reverse transcriptase (Life Technologies). Generation of MS Quantitative PCR (qPCR) was performed using a 7300 Real-time PCR Semiconfluent monolayers cultures were suspended in the complete System and SYBR Green (Life Technologies). Relative expression levels medium and 4 × 104 cells per well were plated in 48-well plates (Corning were calculated as described.46 Hydroxymethylbilane synthase or hypox- Life Sciences, Tewksbury, MA, USA) coated with 1% agarose in RPMI. After anthine guanine phosphoribosyltransferase were used as endogenous 4 days, a single MS spontaneously aggregated in each well. MS were control genes for expression data normalization. readily handled with a sterile Pasteur pipette for subsequent experiments.

Immunohistochemistry Proliferation rates by BrdU nuclear labeling Frozen sections of tumor biopsies were fixed for 30 min in 4% Four-day-old MS were incubated for 6 h at 37 °C in a complete medium μ paraformaldehyde, followed by 5 min denaturation in cold acetone. containing 10 M of BrdU (Pharmingen, San Diego, CA, USA). After this Endogenous peroxidase activity was depleted by incubation with 3% period, MS were dissociated with Trypsin-EDTA and single-cell suspensions hydrogen peroxide solution. Sections were permeabilized for 1 h with 0.1% were fixed, permeabilized and incubated with fluorescein isothiocyanate- conjugated anti-BrdU antibody in accordance with the manufacturer's Triton X-100 and incubated overnight at 4 °C with anti-ADAM23 4 (HPA012130; Sigma-Aldrich Co., St Louis, MO, USA). ADAM23 immunos- protocol (BD Pharmingen). BrdU-labeled nuclei were analyzed in 10 cells taining were visualized using a Tyramide Amplification Kit (T20921; Life per sample on a FACSCalibur cytometer (BD Pharmingen). Technologies) and 3,3′-diaminobenzidine substrate (Vector Laboratories, Burlingame, CA, USA). The tissue sections were counterstained with Clonogenic anchorage-independent assays hematoxylin, dehydrated and coverslipped with Permount (Fisher Scien- μ tific, Waltham, MA, USA). Single-cell suspensions were mixed to 500 l of 0.6% agarose diluted in the complete medium and were plated in culture dishes coated with 5 mm of 1% agarose. Plates were incubated at 37 °C and 5% CO2 for 2 weeks to allow colony formation. The number of colonies formed and the colony Cell lines and cell culture area (mm2) were determined under the light microscope using the ImageJ The MDA-MB-435 tumor cell line was obtained from the ATCC (Manassas, software (NIH, Bethesda, MD, USA). VA, USA), the SK-Mel-37 human melanoma and CMS5a murine sarcoma cell lines were donated by Dr LJ Old and by Dr G Ritter (Memorial Sloan- Kettering Cancer Centre, New York, NY, USA). All cells were maintained at Subcutaneous tumorigenesis assays 6 μ 37 °C, under 5% CO2, in a complete medium: RPMI 1640 (Life Technologies) In all, 10 tumor cells suspended in 100 l of phosphate-buffered saline supplemented with 10% fetal bovine serum (Cultilab, Campinas, SP, Brazil) were inoculated subcutaneously in the flank of immunodeficient female – and 1% L-glutamine (Sigma-Aldrich Co.). BALB/c nude mice (4 10 weeks old). Tumor growth was monitored weekly and tumor sizes were determined by measuring two perpendicular tumor diameters through the tumor long axis using a caliper. All experiments were conducted according to ethic standards and approved by the Animal Ablation of ADAM23, LGI4 and NOS3 genes by shRNAs and Experimentation Ethics Committee of the Medical and Research Centre at ADAM23 overexpression Hospital AC Camargo (043/09). shRNA ADAM23 ablation was performed as described previously.27 For the sake of simplicity, results obtained for the wild-type cells and cells transfected with a control shRNA vector were grouped throughout the Invasion assays into three-dimensional collagen matrices text and presented as the A23pos, whereas results obtained for two MS were embedded in 1.5–3 mg/ml of reconstituted rat tail type-1 independent ADAM23-shRNA clones (shA23 nos. 1 and 2) were grouped collagen (BD Pharmingen) and plated in 48-well plates coated with 5 mm and presented as A23neg. LGI4 (shLGI4) and NOS3 (shNOS3) ablations of 1% agarose. After polymerization, collagen matrices were overlaid with were obtained by lentiviral infection using pLKO.1 vector containing two the complete medium. Average invasion speed was obtained dividing the different shRNA sequences obtained from Sigma-Aldrich Co. shRNAs to distances, from the MS border, traveled by ‘leader’ cells (80–100 tumor GFP sequence was used as control (shCtrl) (Sigma-Aldrich Co.). Viral cells per spheroid) by time. Invasion was determined under the bright field production was carried out using Lipofectamine-mediated transfection of or fluorescence microscopy using the ImageJ software (NIH). human embryonic kidney 293T cells. CMS5a cells were transfected with pcDNA3.1 empty vector or pcDNA3.1-ADAM23-HA (splicing isoform alpha) kindly donated by Dr S Cal.47 Spontaneous metastasis assay Metastatic assays were performed as described previously.48 ADAM23- heterotypic tumors were generated by inoculation of a single-cell suspension of A23pos and A23neg cells at a 1:1 ratio. Primary tumors were Membrane isolation protocol surgically removed when they reached a mean diameter of 1 cm tumors A total of 5 × 106 cells were washed with ice-cold phosphate-buffered and skin incisions were closed. After 8-week interval, animals were killed saline and scraped into the membrane extraction buffer (50 mM Tris–HCl and autopsied. These experiments were conducted according to ethic (pH 7.4), 10% sucrose and 5 mM EDTA) containing protease inhibitors and standards and approved by the Animal Experimentation Ethics Committee were incubated at 4 °C under agitation for 30 min. Cells were disrupted by from FAP– AC Camargo (043/09). passing through a 27G needle and cell membranes were precipitated by centrifugation at 18 000 g for 75 min. Pellets were suspended in NP40 buffer (20 mM Tris–HCl (pH 8.0), 137 mM NaCl, 10% glycerol, 1% Nonidet Haptotactic migration assay P-40 and 2 mM EDTA) containing protease inhibitors for 6 h and cleaned at Migration assays were carried out using transwell plates (Corning Life 18 000 g for 30 min. Sciences) with 8 μm polycarbonate filters precoated with 20 μg/ml rat tail

Oncogene (2014), 1 – 10 © 2014 Macmillan Publishers Limited ADAM23-ITH promotes tumor growth and metastasis ET Costa et al 9 collagen type-1 (BD Pharmingen). A total of 4 × 104 cells were plated in the 18 Yachida S, Jones S, Bozic I, Antal T, Leary R, Fu B et al. Distant metastasis occurs upper chamber of the transwell and allowed to migrate for 8 h at 37 °C. late during the genetic evolution of pancreatic cancer. Nature 2010; 467: At the end of the experiment, the top side of the filters were scraped 1114–1119. with cotton swabs and migrating cells were fixed and stained with 19 Yancovitz M, Litterman A, Yoon J, Ng E, Shapiro RL, Berman RS et al. Intra- and 4′,6-diamidino-2-phenylindole (Sigma-Aldrich Co.). Cells were counted at inter-tumor heterogeneity of BRAFV600E mutations in primary and metastatic x100 magnification in 20 different optical fields/insert. melanoma. PLoS One 2012; 7: e29336. 20 Almendro V, Marusyk A, Polyak K. 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ADAMs: key components in EGFR signalling and development. Nat Rev Mol Cell Biol 2005; 6:32–34. CONFLICT OF INTEREST 25 Costa FF, Colin C, Shinjo SM, Zanata SM, Marie SK, Sogayar MC et al. ADAM23 The authors declare no conflict of interest. methylation and expression analysis in brain tumors. Neurosci Lett 2005; 380: 260–264. 26 Costa FF, Verbisck NV, Salim AC, Ierardi DF, Pires LC, Sasahara RM et al. Epigenetic silencing of the adhesion molecule ADAM23 is highly frequent in breast tumors. ACKNOWLEDGEMENTS Oncogene 2004; 23:1481–1488. We are grateful to Dr Andrew Simpson and Dr Raphael Parmigiani for critical reading 27 Verbisck NV, Costa ET, Costa FF, Cavalher FP, Costa MD, Muras A et al. ADAM23 of this manuscript and to Dr Rodrigo Perez, Dr Alicia Kowaltowski, Dr Beatriz negatively modulates alpha(v)beta(3) integrin activation during metastasis. Geronymo, Dr Camila Machado, Dr Vilma Martins, Dr Tiago dos Santos and Dr Anibal Cancer Res 2009; 69: 5546–5552. 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Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc)