Oncogene (2016) 35, 2777–2788 © 2016 Macmillan Publishers Limited All rights reserved 0950-9232/16 www.nature.com/onc ORIGINAL ARTICLE Loss of the tumor suppressor spinophilin (PPP1R9B) increases the cancer stem cell population in breast tumors I Ferrer1, EM Verdugo-Sivianes1, MA Castilla1, R Melendez1, JJ Marin1,2, S Muñoz-Galvan1, JL Lopez-Guerra3, B Vieites4, MJ Ortiz-Gordillo3, JM De León5, JM Praena-Fernandez6, M Perez1, J Palacios7 and A Carnero1,8 The spinophilin (Spn, PPP1R9B) gene is located at 17q21.33, a region frequently associated with microsatellite instability and loss of heterozygosity, especially in breast tumors. Spn is a regulatory subunit of phosphatase1a (PP1), which targets the catalytic subunit to distinct subcellular locations. Spn downregulation reduces PPP1CA activity against the retinoblastoma protein, pRb, thereby maintaining higher levels of phosphorylated pRb. This effect contributes to an increase in the tumorigenic properties of cells in certain contexts. Here, we explored the mechanism of how Spn downregulation contributes to the malignant phenotype and poor prognosis in breast tumors and found an increase in the stemness phenotype. Analysis of human breast tumors showed that Spn mRNA and protein are reduced or lost in 15% of carcinomas, correlating with a worse prognosis, a more aggressive tumor phenotype and triple-negative tumors, whereas luminal tumors showed high Spn levels. Downregulation of Spn by shRNA increased the stemness properties along with the expression of stem-related genes (Sox2, KLF4, Nanog and OCT4), whereas ectopic overexpression of Spn cDNA reduced these properties. Breast tumor stem cells appeared to have low levels of Spn mRNA, and Spn loss correlated with increased stem-like cell appearance in breast tumors as indicated by an increase in CD44+/CD24- cells. A reduction of the levels of PPP1CA mimicked the cancer stem-like cell phenotype of Spn downregulation, suggesting that the mechanism of Spn involves PP1a. These increased cancer stem cell-like properties with reduced Spn might account for the malignant phenotype observed in Spn-loss tumors and may contribute to a worse patient prognosis. Oncogene (2016) 35, 2777–2788; doi:10.1038/onc.2015.341; published online 21 September 2015 INTRODUCTION colorectal, gastric, renal and lung carcinomas, as well as in salivary Spinophilin (Spn, also known as neurabin II and PPP1R9B) was first gland carcinosarcoma, an extremely aggressive neoplasm. isolated independently by two laboratories and has been shown In extensive LOH mapping in primary lung carcinoma using 15 17 to interact with protein phosphatase 1 (PP1) and F-actin.1,2 The highly polymorphic markers, the highest LOH value appeared domain structure of Spn suggests that it functions as a with the D17S588 marker, showing a 53% loss. This marker multifunctional protein scaffold that regulates protein–protein is located exactly within the Spn locus. However, the neighboring interactions.3,4 More than 30 partner proteins of Spn have been tumor suppressor genes, such as BRCA1, were not significantly discovered3,5–7 to impinge on both membrane and cytoskeletal affected (6–13% LOH). Deep analysis of human lung tumors functions.3,8 However, the physiological relevance of some showed that the Spn protein is absent in 20% of neoplasias and of these interactions remains to be determined. Spn performs reduced in another 38%. Spn reduction correlates with malignant important functions in the nervous system where it has been grade and p53 mutations.18 Low Spn levels have been associated implicated in the regulation of spine morphology and density, with a high proliferative grade and poor patient prognosis in synaptic plasticity and neuronal migration.9,10 hepatocarcinoma, head and neck cancer and advanced stages of – The Spn gene is located at 17q21.33, a cytogenetic area colorectal carcinoma.19 21 frequently associated with microsatellite instability and loss At a functional level, Spn regulates PP1 activity,22 whereas loss of heterozygosity (LOH). Most of the studies on the 17q21 locus of Spn reduces PPP1CA activity on its target pRb,23 thereby have focused on BRCA1, which exhibits a variable frequency in maintaining higher levels of phosphorylated pRb.24 In the absence LOH depending on the type and stage of the tumor.4 However, of p53, reduced levels of Spn increase the tumorigenic properties several studies have suggested the presence of another tumor of cells.24 Spn-KO mice have a reduced lifespan, an increased suppressor gene in this area that includes the Spn locus,11–16 number of tumors and increased cellular proliferation in some which indicates the importance of this locus independent tissues, such as the mammary ducts. In addition, the combined of BRCA1 in the pathology of cancer, especially in breast tumors loss of Spn and p53 activity in mouse models leads to an increase (Supplementary Table I). LOH in chromosome 17q21.33 has been in mammary carcinomas, which confirms the functional relation- observed in different tumors, including breast, ovarian, prostate, ship between p53 and Spn.25 1Instituto de Biomedicina de Sevilla (IBIS/HUVR/CSIC/Universidad de Sevilla), Seville, Spain; 2Department of Preventive Medicine and Public Health, Seville University, Seville, Spain; 3Department of Radiation Oncology, University Hospital Virgen Del Rocío, Seville, Spain; 4Department of Pathology, University Hospital Virgen Del Rocío, Seville, Spain; 5Unidad Funcional de Patología Mamaria, University Hospital Virgen Del Rocío, Seville, Spain; 6Methodology Unit- Fundación Pública Andaluza para la Gestión de la Investigación en Salud de Sevilla, Virgen del Rocío University Hospital, Seville, Spain; 7Department of Pathology, University Hospital Ramon y Cajal, Madrid, Spain and 8Consejo Superior de Investigaciones Cientificas, Sevilla, Spain. Correspondence: Professor A Carnero, Instituto de Biomedicina de Sevilla (IBIS/HUVR/CSIC/US), Campus HUVR, Edificio IBIS, Avda. Manuel Siurot s/n, Sevilla 41013, Spain. E-mail: [email protected] Received 18 December 2014; revised 6 August 2015; accepted 7 August 2015; published online 21 September 2015 Loss of Spn increases the cancer stem cell population in breast tumors I Ferrer et al 2778 The pRb pathway controls several aspects of stem cell biology, obtained from prophylactic surgery. We found that 80–85% of the including the tight control of self-renewal characteristics of tumors displayed high levels of Spn, similar to the values observed progenitor cells.26,27 It is tempting to speculate that the loss in normal glands (Figures 1a and b and Supplementary Figure S1). of Spn may result in a partial expansion of certain progenitor Another 10–15% of tumors showed intermediate levels of Spn pools. This expansion may explain the preneoplastic abnormalities (Figures 1a and b). In addition, 2.4% of tumors showed no associated with increased cell proliferation found in Spn-null Spn expression (Figures 1a and b). We also observed that loss of 24,25 mice. Activation of a p53-mediated arrest may further limit Spn correlated with a higher histological grade determined at the expansion of these cells. Elimination of this additional barrier may time of surgery (Figure 1c) and a less differentiated phenotype therefore explain the synergistic effect observed in terms of tumor (Figure 1d). formation in Spn-null animals when p53 function is impaired. Collectively, our data suggest that Spn is lost in a small Furthermore, Spn can restrain self renewal of brain tumor-initiating percentage of human breast tumors at different extents and that 28 29 cells and anchorage-independent growth of glioma cell lines. lower or null levels of Spn may correlate with a higher In the present work, we have attempted to understand the Spn histological grade. loss-induced causal effect in cancer, specifically in the breast, given the relevance of its locus, which might explain the relevance of its loss during tumorigenesis. We found that the Spn protein is Loss of Spn correlates with worse prognosis and the triple- reduced or lost in 15% of breast carcinomas, which correlates with negative phenotype in breast tumors a more aggressive tumor phenotype. Furthermore, we have To explore the relevance of Spn loss in breast tumors, observed an inverse correlation between Spn and cancer stem we extracted mRNA from tumor samples from a second cohort cells (CSCs, also called cancer-initiating cells). The enforced of human breast tumors comprising different types of tumors. downregulation of Spn by shRNA induced CSC-like properties in The analysis of the levels of mRNA indicates that Spn levels were breast tumor cells accompanied by an increase in stem factors. especially reduced in ER-negative tumors (Student’s t-test We also observed a significant correlation of Spn loss with triple- Po0.001; Figure 2a) and in triple-negative tumors in contrast to negative breast tumors, whereas luminal tumors maintain higher the luminal type tumors (Student’s t-test Po0.001; Figure 2b). levels of Spn mRNA. Therefore, the increased CSC-like properties These data indicate that reduced Spn levels are more likely induced by the downregulation of Spn might account for the to occur in breast tumors with a worse prognosis. There was no increased malignant phenotype observed in Spn-null breast difference in Spn levels between luminal A or B type tumors tumors and its correlation with a poor prognosis. (Figure 2b). Specific analysis of the population of Her2-positive (ER and PR negative) tumors showed that these tumors exhibited lower levels of Spn mRNA (Figure 2c). Similar data were obtained RESULTS from the analysis of cohort 1 by immunohistochemistry Analysis of Spn in human breast tumors (Supplementary Table S4). To study whether reductions in Spn levels are a common event To confirm the effect of Spn loss on patient prognosis, we in human breast tumors, we generated a tissue array of cohort 1 analyzed disease-free survival in cohort 1, for which we have human breast tumors at different stages and different histo- clinical follow-up.