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King’s Research Portal DOI: 10.3892/ijo.2016.3521 Document Version Peer reviewed version Link to publication record in King's Research Portal Citation for published version (APA): Woodman, N., Pinder, S. E., Tajadura, V., Le Bourhis, X., Gillett, C., Delannoy, P., Burchell, J. M., & Julien, S. (2016). Two E-selectin ligands, BST-2 and LGALS3BP, predict metastasis and poor survival of ER-negative breast cancer. International Journal of Oncology, 49(1), 265-275. https://doi.org/10.3892/ijo.2016.3521 Citing this paper Please note that where the full-text provided on King's Research Portal is the Author Accepted Manuscript or Post-Print version this may differ from the final Published version. If citing, it is advised that you check and use the publisher's definitive version for pagination, volume/issue, and date of publication details. 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Download date: 02. Oct. 2021 INTERNATIONAL JOURNAL OF ONCOLOGY 49: 265-275, 2016 Two E-selectin ligands, BST-2 and LGALS3BP, predict metastasis and poor survival of ER-negative breast cancer Natalie WOODMAN1, SARAH E. PINDER1, VIRGINIA TAJADURA2, XUEFEN LE BOURHIS3, Cheryl GILLETT1, PHILIPPE DELANNOY4, JOY M. BURCHELL2 and Sylvain JULIEN3,4 1Breast Research Pathology, 2Breast Cancer Biology, Research Oncology, King's College London, Guy's Hospital, London, UK; 3University of Lille, INSERM, U908 - CPAC, Cell Plasticity and Cancer; 4University of Lille, CNRS, UMR 8576 - UGSF, Unit of Structural and Functional Glycobiology, Lille, Villeneuve d'Ascq, France Received October 5, 2015; Accepted January 11, 2016 DOI: 10.3892/ijo.2016.3521 Abstract. Distant metastases account for the majority of breast cancers. We found both glycoproteins to be associated cancer-related deaths in breast cancer. The rate and site of with distant metastasis risk and poorer survival. Importantly, metastasis differ between estrogen receptor (ER)-negative concomitant high expression of BST-2 with sLex defined a sub- and ER-positive tumours, and metastatic fate can be very group of patients with ER-negative tumours displaying higher diverse even within the ER-negative group. Characterisation risks of liver and brain metastasis and a 3-fold decreased of new pro-metastatic markers may help to identify patients survival rate. with higher risk and improve their care accordingly. Selectin ligands aberrantly expressed by cancer cells promote metas- Introduction tasis by enabling interaction between circulating tumour cells and endothelial cells in distant organs. These ligands consist The 5-year overall survival of breast cancer patients with distant in carbohydrate molecules, such as sialyl-Lewis x antigen metastasis is 23.4% (http://seer.cancer.gov/ for details see ref. 1) (sLex), borne by glycoproteins or glycolipids on the cancer cell and once detected, metastatic breast cancer is incurable. Distant surface. We have previously demonstrated that the molecular metastases arise from circulating cancer cells that migrate from scaffold presenting sLex to selectins (e.g. glycolipid vs. glyco- the blood stream to colonise a distant organ (extravasation) (2). proteins) was crucial for these interactions to occur. Moreover, Specific inhibition of cancer cell extravasation would therefore we reported that detection of sLex alone in breast carcinomas potentially lead to a decrease in the incidence of metastasis. was only of limited prognostic value. However, since sLex was Thus, delineating the molecular mechanisms involved in found to be carried by several glycoproteins in cancer cells, this process is one of the crucial challenges for breast cancer we hypothesized that the combination of the carbohydrate treatment. with its carriers could be more relevant than each marker One such mechanism is the interaction between sialyl- independently. In this study, we addressed this question by Lewis x (sLex) antigen expressed by cancer cells and the analysing sLex expression together with two glycoproteins selectins expressed by endothelial or circulating immune (BST-2 and LGALS3BP), shown to interact with E-selectin in cells (3). There is a compelling body of evidence that a carbohydrate-dependent manner, in a cohort of 249 invasive sLex/selectin interaction is involved in metastatic progression in several types of solid tumours including gastric (4), lung (5) and prostate (6) cancers. However, the relationship in breast cancer metastasis remains controversial (7). Correspondence to: Dr Sylvain Julien, Cell Plasticity and Cancer, We have previously reported that sLex was over-expressed Inserm U908, Batiment SN3, Université, Lille 1, 59655 Villeneuve in estrogen receptor (ER)-negative breast tumours compared to d'Ascq, France ER-positive ones. Although ER-negative cancers are known to E-mail: [email protected] typically be of higher grade and intrinsically more aggressive and more often develop metastatic foci, we found no evidence Abbreviations: BCSS, breast cancer specific survival; BST-2, that sLex was correlated with metastatic behaviour either in the bone marrow stromal antigen 2; DM, distant metastasis; ER, estrogen receptor; HUVECs, human umbilical vein endothelial ER-negative or the ER-positive group (7). cells; LGALS3BP, galectin-3-binding protein; LN, lymph node; This lack of association led us to question the context of x mAb, monoclonal antibody; NHS, national health service; PR, expression of sLe in breast cancer cells, asking whether the x progesterone receptor; sLex, sialyl-Lewis x antigen; TMA, tissue scaffold carrying sLe (e.g. glycosphingolipids or specific microarray glycoproteins) influenced its role in metastasis. Indeed, we reported that rolling on endothelial cells was more efficient Key words: breast cancer, sialyl-Lewis x, BST-2, LGALS3BP, when sLex was carried by glycoproteins rather than glyco- metastasis lipids (7). However, sLex was found to be carried by several membrane bound glycoproteins in a breast cancer cell line that 266 woodman et al: Two glycoproteins predict metastasis and survival in ER-negative breast cancer displayed selectin-dependent rolling on endothelial lining. We Table I. Clinical and biological features of the cohort of identified two of these glycoproteins as tetherin, also known 249 patients. as bone marrow stromal antigen 2 (BST-2), and the galectin- 3-binding protein (also named MAC-2BP) encoded by the Characteristics Data LGALS3BP gene (7). Subsequently, Shirure et al have shown that MAC-2BP carrying sLex was functionally involved in Median age at diagnosis: year 57.0 (Min: 29.0, Max 94.0) ZR-75-1 rolling on HUVECs cultured as monolayer (8). Average tumour size: cm 2.64 x In the present study, we assessed the expression of sLe , Histological grade: n (%) BST-2 and LGALS3BP in a cohort of 249 invasive breast G1 41 (16.5) cancer patients. We investigated whether the combination of sLex with one or other of its known carriers has a bearing on G2 93 (37.3) the prognostic value of sLex. G3 97 (39.0) ND 18 (7.2) Materials and methods Lymph node Involvement: n (%) Positive 128 (51.4) Human samples. The samples used in the study were provided Negative 108 (43.4) by the King's Health Partners Cancer Biobank under their generic ethics approval from National Research Ethics ND 13 (5.2) Service, Committee East of England-Cambridge East, refer- Estradiol receptor (ER): n (%) ence 12/EE/0493. Under this approval the Biobank are also Positive 189 (75.9) able to provide pseudo-anonymised samples for research Negative 57 (22.9) without informed consent if they were collected before ND 3 (1.2) September 2006. All tissue samples used in the study were collected prior to Human Tissue Act 2004. Progesterone receptor (PR): n (%) Positive 153 (61.4) Tissue microarray (TMA) construction and analysis. Negative 93 (37.3) Formalin-fixed paraffin wax embedded tissue from consecu- ND 3 (1.2) tive cases of invasive breast carcinoma dating from 1990 to HER2: n (%) 1995 (n=400) were selected from the King's Health Partners Cancer Biobank. Clinical data, including date and site of Positive 110 (44.2) metastatic recurrence, were collected and validated for each Negative 25 (10.0) sample. From each block 0.6-mm diameter cores were marked ND 114 (45.8) on haematoxylin and eosin-stained slides and the invasive Median follow-up: months 148.0 cancer subsequently sampled using the Beecher TMArrayer (Wisconsin, MA, USA) and placed into replicate, 100 core Local recurrences: n (%) 37 (14.9) TMA blocks. Sections from two duplicate TMA blocks were Distant recurrences: n (%) 73 (29.3) cut at 3 µm and dried overnight at 37˚C. Sections were baked Breast cancer specific death: n (%) 68 (27.3) for 2 h at 60˚C, blocked using 10% bovine serum albumin x in PBS and stained with anti-sLe monoclonal antibody ND, not documented. (mAb) HECA-452 (7) (BD Biosciences, Oxford, UK) at a concentration of 1 µg.ml-1, anti-LGALS3PB mAb 6B7 (Novus Biologicals, Cambridge, UK) at a concentration of 0.2 µg.ml-1, or anti-BST-2 polyclonal antibody (9) (Novus Biologicals) at a concentration of 0.2 µg.ml-1.