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Evaluation of Lumican Effects on Morphology of Invading Breast Evaluation of lumican effects on morphology of invading breast cancer cells, expression of integrins and downstream signaling Konstantina Karamanou, Marco Franchi, Maurizio Onisto, Alberto Passi, Demitrios Vynios, Stéphane Brézillon To cite this version: Konstantina Karamanou, Marco Franchi, Maurizio Onisto, Alberto Passi, Demitrios Vynios, et al.. Evaluation of lumican effects on morphology of invading breast cancer cells, expression of integrins and downstream signaling. FEBS Journal, Wiley, 2020, 287, pp.4862 - 4880. 10.1111/febs.15289. hal-02986565 HAL Id: hal-02986565 https://hal.univ-reims.fr/hal-02986565 Submitted on 17 Nov 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Distributed under a Creative Commons Attribution| 4.0 International License Evaluation of lumican effects on morphology of invading breast cancer cells, expression of integrins and downstream signaling Konstantina Karamanou1,2,3 , Marco Franchi4 , Maurizio Onisto5 , Alberto Passi6 , Demitrios H. Vynios1 and Stephane Brezillon 2,3 1 Biochemistry, Biochemical Analysis & Matrix Pathobiology Research Group, Laboratory of Biochemistry, Department of Chemistry, University of Patras, Greece 2 Laboratoire de Biochimie Medicale et Biologie Moleculaire, Universite de Reims Champagne-Ardenne, France 3 Matrice Extracellulaire et Dynamique Cellulaire, CNRS UMR 7369, Reims, France 4 Department for Life Quality Studies, University of Bologna, Rimini, Italy 5 Department of Biomedical Sciences, University of Padua, Italy 6 Department of Surgical and Morphological Sciences, University of Insubria, Varese, Italy Keywords The small leucine-rich proteoglycan lumican regulates estrogen receptors breast cancer; integrins; invadopodia; (ERs)-associated functional properties of breast cancer cells, expression of lumican; proteoglycans matrix macromolecules, and epithelial-to-mesenchymal transition. How- ever, it is not known whether the ER-dependent lumican effects on breast Correspondence S. Brezillon, Matrice Extracellulaire et cancer cells are related to the expression of integrins and their intracellular Dynamique Cellulaire (MEDyC), CNRS UMR signaling pathways. Here, we analyzed the effects of lumican in three n°7369, Universite de Reims Champagne- breast cancer cell lines: the highly metastatic ERb-positive MDA-MB-231, Ardenne, 51 rue Cognacq Jay, Reims cells with the respective ERb-suppressed (shERbMDA-MB-231), and lowly 51095, France invasive ERa-positive MCF-7/c breast cancer cells. Scanning electron Tel: + 33 3 26 91 37 34 microscopy, confocal microscopy, real-time PCR, western blot, and cell E-mail: [email protected] adhesion assays were performed. Lumican effects on breast cancer cell (Received 6 June 2019, revised 11 October morphology were also investigated in 3-dimensional collagen cultures. 2019, accepted 9 March 2020) Lumican treatment induced cell–cell contacts and cell grouping and inhib- ited microvesicles and microvilli formation. The expression of the cell sur- doi:10.1111/febs.15289 face adhesion receptor CD44, its isoform and variants, hyaluronan (HA), and HA synthases was also investigated. Lumican inhibited the expression of CD44 and HA synthases, and its effect on cell adhesion revealed a major role of a1, a2, a3, aVb3, and aVb5 integrins in MDA-MB-231 cells, but not in MCF-7/c cells. Lumican upregulated the expression of a2 and b1 integrin subunits both in MDA-MB-231 and in shERbMDA-MB-231 as compared to MCF-7/c cells. Downstream signaling pathways for inte- grins, such as FAK, ERK 1/2 MAPK 42/44, and Akt, were found to be downregulated by lumican. Our data shed light to the molecular mecha- nisms responsible for the anticancer activity of lumican in invasive breast cancer. Abbreviations As, anti-sense; CD44, cluster of differentiation 44; CD44s, CD44 (standard isoform); ECM, extracellular matrix; EGFR, epidermal growth factor receptor; EMT, epithelial-to-mesenchymal transition; ERK, extracellular-related kinase; FAK, focal adhesion kinase; HA, hyaluronic acid; KS, keratan sulfate; LRR, leucine-rich repeat; MAPK, mitogen-activated protein kinases; MMP, matrix metalloproteinase; MT1-MMP, membrane type 1–matrix metalloproteinase; PG, proteoglycan; SLRP, small leucine-rich proteoglycans. The FEBS Journal (2020) ª 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of 1 Federation of European Biochemical Societies This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. Lumican effect on breast cancer cell phenotype K. Karamanou et al. Introduction characterized as tightly packed, organelle-free, actin- rich protrusions of the plasma membrane, playing a Breast cancer is a major research field, as it is the pre- governing role in the steps of metastasis, from invasion dominant type of cancer among women with 25% of to the neighboring stroma, intravasation, and finally cancer cases to be identified as breast cancer incidents extravasation [13,14]. Cytoskeletal changes at invadopo- [1]. A great hallmark of breast cancer is the absence or dia are monitored by cortactin, and extracellular matrix presence of estrogen receptors alpha and beta (ERa (ECM)-degrading function of invadopodia is deter- and ERb). Estrogens are a hormonally active family, mined by membrane type-1–matrix metalloproteinase having a major role in several cellular functions of (MT1-MMP or MMP-14); therefore, cortactin and breast cancer and being related to the expression of MMP-14 are two major proteins governing the matrix macromolecules, and therefore with the cell–cell invadopodia formation. Cortactin is reported to be and cell–extracellular matrix interactions [2,3]. There overexpressed by 13% of breast carcinomas, and its are two different estrogen receptors, in terms of genet- overexpression enhances cell migration, invasion, and ics, structure, and, therefore, functionality, the ERa tumor cell metastasis, whereas when it is blocked, the and ERb [4].ERa is considered to be the most impor- Src signaling is thereafter blocked, leading to the inhibi- tant subtype in the mammary epithelium, and there- tion of invadopodia formation [15-20]. It is also worth fore, it is considered as a prognostic marker for breast noticing that cortactin is essential for the maintenance cancer incidents. ERa is fertile ground for pharmaceu- of F-actin-enriched invadopodia core structures, as well tical targeting of breast cancer, since the 17b-estradiol as for the initiation and sustainability of matrix degra- (E2)/estrogen receptor alpha (ERa) signaling is of piv- dation, in cooperation with MMP-14. Integrins are con- otal importance, as it features 70% of breast cancer sidered to bind and activate MMPs, through which they cases as ERa-positive [5]. Although most ERa-positive regulate the effective invasion of invadopodia into the tumors respond initially to anti-estrogenic therapies, extracellular matrix. It has been observed that b1 inte- that is, tamoxifen, they end up resistant independently grin coclusters with MMP-14 at sites of interaction with of the ER status and they potentially result in endome- collagen fibers along the cell leading edge, and therefore, trial carcinoma [6]. Therefore, it is regarded as funda- the adhesive mechanism is involved in invadopodia mat- mental to investigate new therapies, with less side uration and ECM degradation [21]. effects and higher specificity to the target cell. Vinculin is a governing protein in several cell func- Although the biological activity of ERa is exten- tions, such as cell adhesion, cell migration, and embry- sively explored, its ERb isoform is less investigated. It onic development, since it has the ability to interact is reported that when ERb is being activated, it poten- with integrins to the cytoskeleton at the focal adhe- tially forms heterodimers with ERa, and therefore sions [22-25]. Therefore, vinculin is a fundamental affects the biological functions of ERa, too [7,8].It marker of cell–cell junctions and focal adhesion, was recently reported that the triple-negative, but located in the periphery of newly created invadopodia. ERb-positive, MDA-MB-231 breast cancer cells [9,10], Consequently, the downregulation of vinculin prevents when transfected with shRNA against human ERb, or inhibits several cellular functions, such as cell adhe- ERb mRNA was suppressed by 70% triggering major sion, formation of focal adhesions, as well as lamel- alterations in cell functions of the aggressive breast lipodia protrusions [26], whereas its overexpression cancer cells, as well as in the epithelial-to-mesenchymal facilitates cell adhesion and recruitment of cytoskeletal (EMT) markers, leading to a potent mesenchymal-to- proteins at the domains of integrin binding at the focal epithelial transition (MET) situation. The ERb-sup- adhesions [27,28]. pressed MDA-MB-231 cells present an epithelial-like
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