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The Expression of the Zonula Adhaerens Protein PLEKHA7 Is Strongly Decreased in High Grade Ductal and Lobular Breast Carcinomas

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The Expression of the Zonula Adhaerens Protein PLEKHA7 Is Strongly Decreased in High Grade Ductal and Lobular Breast Carcinomas RESEARCH ARTICLE The Expression of the Zonula Adhaerens Protein PLEKHA7 Is Strongly Decreased in High Grade Ductal and Lobular Breast Carcinomas Jean-Christophe Tille1, Liza Ho1, Jimit Shah2,3, Olivia Seyde1, Thomas A. McKee1, Sandra Citi2,3* 1 Division of Clinical Pathology, Geneva University Hospitals, Geneva, Switzerland, 2 Department of Cell Biology, University of Geneva, Geneva, Switzerland, 3 Institute of Genomics and Genetics of Geneva (iGE3), University of Geneva, Geneva, Switzerland * [email protected] Abstract OPEN ACCESS PLEKHA7 is a junctional protein, which participates in a complex that stabilizes E-cadherin at the zonula adhaerens. Since E-cadherin is involved in epithelial morphogenesis, signal- Citation: Tille J-C, Ho L, Shah J, Seyde O, McKee ing, and tumor progression, we explored PLEKHA7 expression in cancer. PLEKHA7 TA, Citi S (2015) The Expression of the Zonula Adhaerens Protein PLEKHA7 Is Strongly Decreased expression was assessed in invasive ductal and lobular carcinomas of the breast by immu- in High Grade Ductal and Lobular Breast nohistochemistry, immunofluorescence and quantitative RT-PCR. PLEKHA7 was detected Carcinomas. PLoS ONE 10(8): e0135442. at epithelial junctions of normal mammary ducts and lobules, and of tubular and micropapil- doi:10.1371/journal.pone.0135442 lary structures within G1 and G2 ductal carcinomas. At these junctions, the localization of Editor: Fernando Schmitt, University of Toronto, PLEKHA7 was along the circumferential belt (zonula adhaerens), and only partially overlap- CANADA ping with that of E-cadherin, p120ctn and ZO-1, as shown previously in rodent tissues. PLE- Received: March 18, 2015 KHA7 immunolabeling was strongly decreased in G3 ductal carcinomas and undetectable Accepted: July 22, 2015 in lobular carcinomas. PLEKHA7 mRNA was detected in both ductal and lobular carcino- Published: August 13, 2015 mas, with no observed correlation between mRNA levels and tumor type or grade. In sum- mary, PLEKHA7 is a junctional marker of epithelial cells within tubular structures both in Copyright: © 2015 Tille et al. This is an open access article distributed under the terms of the Creative normal breast tissue and ductal carcinomas, and since PLEKHA7 protein but not mRNA Commons Attribution License, which permits expression is strongly decreased or lost in high grade ductal carcinomas and in lobular car- unrestricted use, distribution, and reproduction in any cinomas, loss of PLEKHA7 is a newly characterized feature of these carcinomas. medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the Swiss Cancer League, KFS-2813-08-2011 (http://www. Introduction krebsliga.ch) (SC); Swiss Fond National, Breast carcinoma is the most common cancer in women both in the developed and non-devel- 3100_135730 (http://www.snf.ch) (SC); and Ligue oped world, and the second leading cause of cancer death in women, after lung cancer. Breast Genevoise contre le Cancer Project n. 1013 (http:// www.lgc.ch) (JCT). The funders had no role in study carcinomas are classified into invasive and non-invasive, based on their infiltrating characteris- design, data collection and analysis, decision to tics, and comprise a range of tumor types, among which ductal carcinomas are the most com- publish, or preparation of the manuscript. mon, and lobular carcinomas represent about 5–10% of the total. Distinctive morphology and PLOS ONE | DOI:10.1371/journal.pone.0135442 August 13, 2015 1/13 PLEKHA7 Expression in Breast Cancer Competing Interests: The authors have declared infiltrative pattern between ductal and lobular carcinomas are useful to make the diagnosis. that no competing interests exist. Furthermore, E-cadherin and p120ctn immunohistochemistry can be used to distinguish duc- tal from lobular carcinomas, since the vast majority of lobular carcinomas fail to express E-cad- herin, while p120ctn shows a cytoplasmic distribution [1–3]. Recent studies confirm that the molecular hallmark of lobular carcinoma compared to ductal carcinoma is the loss of function of E-cadherin, associated with changes in the expression of genes controlling cytoskeleton remodeling, cell adhesion and extra cellular matrix-interaction pathways [4, 5]. Indeed, loss of cell-cell adhesion, as well as enhanced migration and remodeling of cell and tissue architecture, is of fundamental importance in the local invasiveness and metastatic spread of cancer cells [6]. In epithelial tissues cell-cell adhesion relies primarily on zonulae adhaerentes (ZA) and desmosomes, which contain cadherin family adhesion molecules, and are associated with api- cal tight junctions (TJ, or zonulae occludentes) at the epithelial junctional complex [7]. E-cadherin is the major transmembrane molecule of zonulae adhaerentes, and interacts in the cytoplasm with a protein machinery, which connects it to the actin and microtubule cyto- skeletons [8]. The expression of E-cadherin is controlled at the transcriptional and post-tran- scriptional levels. For example, loss of function of the E-cadherin gene in breast lobular carcinomas has been reported to be due to gene promoter methylation, mutation, and allelic loss [9]. Another mechanism of regulation of E-cadherin expression and function is the control of its retention at the cell surface. The cytoplasmic protein p120ctn binds to the E-cadherin juxta-membrane domain, thus preventing E-cadherin endocytosis, and stabilizing it at the cell surface. Consistently, the loss, mislocalization, aberrant activity or specific isoform expression of p120ctn may be linked to poor tumor prognosis, through the modulation of E-cadherin internalization [10–12]. E-cadherin was recently shown to be stabilized at epithelial junctions by a protein complex that links it to the microtubule cytoskeleton, and which includes p120ctn, paracingulin, and the recently discovered protein PLEKHA7 [13–15]. PLEKHA7 interacts not only with p120ctn and paracingulin, but also with the adherens junction protein afadin [16] and the microtubule minus-end capping protein nezha [13]. In addition, it forms a complex with the TJ proteins cingulin and ZO-1 [17]. Importantly, unlike E-cadherin and associated catenins (p120ctn, α- catenin and β-catenin), but similarly to paracingulin and afadin, PLEKHA7 is not distributed along lateral contacts (puncta adhaerentia) of polarized epithelial cells, but exclusively at cir- cumferential apical zonulae adhaerentes (ZA) [14]. Indeed, the “zonular”, belt-like distribution of PLEKHA7 is remarkably similar to, but not completely overlapping with, the localization of the TJ markers ZO-1 and cingulin [14]. This observation suggests that stabilization of E-cad- herin by the microtubule cytoskeleton occurs only at the ZA, in a specific molecular environ- ment that may also require neighbouring TJ proteins. Of note, genetic studies have implicated PLEKHA7 in hypertension [18, 19] and primary angle closure glaucoma [20]. Moreover, depletion studies in zebrafish embryos indicate a role of PLEKHA7 in cardiac morphogenesis and contractility [21]. Therefore, PLEKHA7 appears to be involved not only in the stabilization of adhesive protein complexes, but also in important physiological and pathological processes. However, the molecular mechanisms implicating PLEKHA7 in organ physiology and pathol- ogy are not clear. Since PLEKHA7 is implicated in the stabilization of E-cadherin complex, and the expression and activities of both E-cadherin and the PLEKHA7-interacting protein p120ctn are linked to tumor formation and metastasis [22], we sought to explore if and how PLEKHA7 expression is altered in epithelial cancer. To this purpose, here we studied the expression and localization of PLEKHA7 in human breast invasive ductal and lobular carcinomas by immunohistochemistry, immunofluorescence, and quantitative RT-PCR. Our results show that PLEKHA7 protein expression and accumulation at sites of cell-cell contact confirm its zonular localization in PLOS ONE | DOI:10.1371/journal.pone.0135442 August 13, 2015 2/13 PLEKHA7 Expression in Breast Cancer human normal and cancer epithelial cells, decreases with increasing grade in ductal breast car- cinomas, and is lost in lobular carcinomas, in a pattern that distinguishes it from other markers of both ZA and TJ. Materials and Methods Tissue samples Formalin fixed paraffin embedded (FFPE) and frozen tissue samples of normal and breast car- cinoma tissues were obtained from the archives of the Division of Clinical Pathology at the Geneva University Hospital, according to a protocol approved by the Geneva Cantonal Com- mittee for Research Ethics (CCER) (Protocol number 14–272, Project title “Expression of junc- tional proteins in breast cancer”), and which has no requirement for written informed consent, because it is a retrospective study. Patient records and information was anonymized and de- identified prior to analysis. Tumor type was assessed according to the World Health Organization criteria [23], grade according to the Nottingham criteria [24], estrogen and progesterone receptor expression was reported with the Allred scoring system, and considered positive if Allred score 3[25], and HER2/neu status (HER2/neu considered amplified if by fluorescence in-situ hybridization a HER2/Cep17 ratio > 2.0). Antibodies The following antibodies were used for immunocytochemistry and/or immunofluorescence: mouse anti-PLEKHA7 (monoclonal 37-8F1 culture
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