Oncogene (2014), 1–10 © 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14 www.nature.com/onc 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 phosphoribosyl-