ANLN is a prognostic biomarker independent of Ki-67 and essential for cell cycle progression in primary breast cancer

Magnusson et al. BMC Cancer (2016) 16:904 DOI 10.1186/s12885-016-2923-8 RESEARCH ARTICLE Open Access ANLN is a prognostic biomarker independent of Ki-67 and essential for cell cycle progression in primary breast cancer Kristina Magnusson1, Gabriela Gremel1, Lisa Rydén2, Victor Pontén1, Mathias Uhlén3, Anna Dimberg1, Karin Jirström4 and Fredrik Pontén1* Abstract Background: Anillin (ANLN), an actin-binding protein required for cytokinesis, has recently been presented as part of a prognostic marker panel in breast cancer. The objective of the current study was to further explore the prognostic and functional value of ANLN as a single biomarker in breast cancer. Methods: Immunohistochemical assessment of ANLN protein expression was performed in two well characterized breast cancer cohorts (n = 484) with long-term clinical follow-up data and the results were further validated at the mRNA level in a publicly available transcriptomics dataset. The functional relevance of ANLN was investigated in two breast cancer cell lines using RNA interference. Results: High nuclear fraction of ANLN in breast tumor cells was significantly associated with large tumor size, high histological grade, high proliferation rate, hormone receptor negative tumors and poor prognosis in both examined cohorts. Multivariable analysis showed that the association between ANLN and survival was significantly independent of age in cohort I and significantly independent of proliferation, as assessed by Ki-67 expression in tumor cells, age, tumor size, ER and PR status, HER2 status and nodal status in cohort II. Analysis of ANLN mRNA expression confirmed that high expression of ANLN was significantly correlated to poor overall survival in breast cancer patients. Consistent with the role of ANLN during cytokinesis, transient knock-down of ANLN protein expression in breast cancer cell lines resulted in an increase of senescent cells and an accumulation of cells in the G2/M phase of the cell cycle with altered cell morphology including large, poly-nucleated cells. Moreover, ANLN siRNA knockdown also resulted in decreased expression of cyclins D1, A2 and B1. Conclusions: ANLN expression in breast cancer cells plays an important role during cell division and a high fraction of nuclear ANLN expression in tumor cells is correlated to poor prognosis in breast cancer patients, independent of Ki-67, tumor size, hormone receptor status, HER2 status, nodal status and age. Keywords: ANLN, Prognostic biomarker, Breast cancer, Proliferation, Antibody-based proteomics Background Breast cancer is the most common female malignancy world-wide and approximately 500 000 women succumb to the disease annually [1]. In Sweden, approximately 9 100 cases of female malignant breast tumors are diagnosed annually. The incidence of breast cancer has * Correspondence: 1 Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden Full list of author information is available at the end of the article shown an annual increase with 1.4% during the last 20 years, at least in part due to an ageing population with increased hormonal replacement therapy and changes in life style, such as obesity and first pregnancy late in life. Furthermore, systematic mammographic screening programs and elevated public awareness have led to the detection of more cases of breast cancer at an early stage. Early detection and a transition to more individualized targeted therapies, has resulted in increased recurrence-free and overall survival rates [2]. Although prognostic gene expression-based profiles have rapidly evolved, there is a need for © The Author(s). 2016 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Magnusson et al. BMC Cancer (2016) 16:904 robust immunohistochemistry (IHC)-based protein biomarkers that can be introduced into clinical praxis. The actin-binding protein ANLN is a ubiquitously expressed protein required for cytokinesis. During the interphase of the cell cycle ANLN is primarily located to the nucleus. At the onset of mitosis, ANLN protein relocates to the cytoplasm where it accumulates in the contractile ring and cleavage furrow during telophase [3]. Recruitment of ANLN to the cleavage furrow is mediated by RhoAdependent mechanisms [4, 5]. Furthermore, ANLN interacts closely with RhoA, stabilizes the localization of the latter to the cleavage furrow and stimulates the expression of active RhoA [4, 6]. Numerous additional proteins, including F-actin, myosin, septins and CD2AP have been shown to interact with ANLN during assembly, maintenance and ingression of the cleavage furrow [7]. Lack of ANLN is generally associated with correct assembly of the cleavage furrow but deficiencies during furrow ingression and completion of cell separation [3, 5]. Consistent with the prominent role of ANLN during cytokinesis, up-regulation of ANLN expression is frequently observed during cancer development, growth and progression [8–10]. It has also been shown that depletion of ANLN expression in human non-small cell lung cancer cells leads to suppression of cell proliferation and an increase of large, poly-nucleated tumor cells [6]. Interestingly, overexpression of the ANLN protein did not only induce cell growth, but also enhanced the migratory capacity of cells, implying a role of ANLN beyond cell cycle control. High ANLN mRNA expression and nuclear ANLN protein expression in lung cancer tissue has been shown to be significantly correlated to poor survival [6, 11]. In another study, cytoplasmic immunoreactivity for ANLN in renal cell carcinomas was associated with a better prognosis, indicating an independent function of ANLN in the cytoplasm [12]. Moreover, ANLN mRNA expression was shown to increase from normal tissue to hyperplasia to malignant and metastatic disease in breast, ovary, renal, colorectal, hepatic, lung, endometrial and pancreatic cancer [8]. The relevance of ANLN protein expression in breast cancer tissue specimens has been explored as a part of a systematic approach to identify novel prognostic biomarkers. O’Leary and co-workers [13] found that a moderate to strong nuclear intensity of ANLN expression was significantly associated with decreased breast cancer specific survival (BCSS) and recurrence free survival (RFS). Using multivariable cox regression analysis, ANLN was suggested as an independent prognostic factor for BCSS following adjustme (...truncated)