Cell Oncol. DOI 10.1007/s13402-015-0226-8

ORIGINAL PAPER

KLF15 in breast cancer: a novel tumor suppressor? Tomomi Yoda 1 & Keely May McNamara 1 & Yasuhiro Miki 1 & Yoshiaki Onodera 1 & Kiyoshi Takagi 2 & Yasuhiro Nakamura 1 & Takanori Ishida 3 & Takashi Suzuki 2 & Noriaki Ohuchi 3 & Hironobu Sasano 1

Accepted: 29 March 2015 # International Society for Cellular Oncology 2015

Abstract Purpose Krüppel-like factor 15 (KLF15) is a transcription factor that is involved in various biological processes, including cellular proliferation, differentiation and death. In addition, KLF15 has recently been implicated in the development of several human malignancies, including breast cancer. In vitro breast cancer studies have pointed at a putative role in the regulation of cell proliferation. As yet, however, KLF15 expression analyses in primary human breast cancers have not been reported. Here, we set out to investigate the clinical and biological significance of KLF15 expression in human breast cancers. Methods KLF15 expression was evaluated by immunohistochemistry in 54 primary invasive ductal breast carcinomas, and its status was correlated with various clinicopathological parameters. We also assessed KLF15 expression in vitro in 4 breast cancer-derived cell lines using Western blotting, and examined the effects of exogenous KLF15 expression on cell cycle progression using flow cytometry. Concomitant (changes in) p21, p27 and TOPO2A expression levels were examined using real-time RT-PCR and immunocytochemistry, respectively. Electronic supplementary material The online version of this article (doi:10.1007/s13402-015-0226-8) contains supplementary material, which is available to authorized users. * Keely May McNamara [email protected] 1

Departments of Pathology, Tohoku University School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan

2

Departments of Pathology and Histotechnology, Tohoku University School of Medicine, Sendai, Japan

3

Department of Surgery, Tohoku University School of Medicine, Sendai, Japan

Results In ~90 % of the primary breast carcinoma tissues tested, KLF15 was found to be expressed and localized in either the cytoplasm, the nucleus or both. Predominant nuclear immunoreactivity was found to be associated with clinicopathological factors predicting a better clinical outcome (i.e., ER positive, HER2 negative, low grade, low Ki-67 expression). The breast cancer-derived cell lines tested showed a low KLF15 expression with a predominant cytoplasmic localization. Subsequent exogenous KLF15 over-expression resulted in a predominant nuclear localization and a concomitant decreased cellular proliferation and an arrest at the G0/G1 phase of the cell cycle. In addition, we found that nuclear KLF15 expression results in up-regulation of p21, a pivotal suppressor of the G1 to S phase transition of the cell cycle. Conclusions Our results indicate that nuclear KLF15 expression suppresses breast cancer cell proliferation at least partially through p21 up-regulation and subsequent cell cycle arrest. This is a first study addressing the role of KLF15 in breast cancer development. Keywords Breast cancer . Cell cycle . Immunohistochemistry . KLF15 . Proliferation

1 Introduction Breast cancer is one of the most common malignancies in women world-wide [1–3]. One of the prognostic biomarkers predicting an adverse clinical outcome of breast cancer patients is a positive Ki-67 expression score [4–6]. Several factors have been reported to regulate cellular proliferation and cell cycle progression. These factors may also affect the proliferation of cancer cells [7, 8]. In order to understand the process of cancer development, including breast cancer, it is important to examine these factors in further detail.

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The Krüppel-like factors (KLFs) constitute a group of transcription factors that contain three different C2H2-type zinc finger domains in their carboxy-terminal regions. These zinc finger domains play important roles in both DNA binding and nuclear localization, allowing KLF family proteins to share common mechanisms of transcription regulation [9, 10]. Several KLF family members have been reported to regulate genes responsible for a variety of biological functions, such as cellular proliferation, differentiation and programmed death (apoptosis). Numerous reports have supported the importance of KLF family members, not only in normal biological processes such as the maintenance of homeostasis, but also in pathological conditions [9, 11]. Accumulating evidence suggests a pivotal role of different KLF family members in human malignancies, including those of the breast, colon, stomach, pancreas and liver [12]. In breast cancer development, important roles for KLF4 and KLF5 have been proposed [13–18]. High KLF5 expression was e.g., found to be associated with an adverse clinical outcome when compared to low expression, and functional analyses have supported tumor-promoting effects of KLF5 in breast carcinoma cells [15–18]. In spite of these results the expression and function of most of the KLF family members in breast cancer development has remained unresolved. Among the KLF family proteins, KLF15, also known as kidney-enriched KLF (KKLF), has been reported to be expressed in multiple tissues, including kidney, liver, heart, adipose and skeletal muscle [19, 20], but its involvement in human malignancies has scarcely been studied. Recent in vitro studies have shown that KLF15 may exert anti-proliferative effects upon various carcinoma cells, including those of the pancreas, endometrium and breast [21, 22]. In the human breast cancer-derived cell line T47D, KLF15 has been reported to decrease estrogen-dependent cell proliferation [22] and to regulate the expression of E2F1, a transcription factor controlling cell cycle progression [23]. These studies suggest a possible regulatory role of KLF15 in breast cancer cell proliferation. As yet, however, the expression and prevalence of KLF15 in primary human breast cancer tissues have not been studied. Therefore, we set out to assess the expression and sub-cellular localization of KLF15 in human breast cancer specimens and to correlate the results with its clinicopathological features. We also assessed the expression and sub-cellular localization of KLF15 in several breast cancer-derived cell lines and assessed their role in proliferation and cell cycle progression.

2 Materials and methods 2.1 Patient samples A total of 54 surgical specimens of invasive ductal carcinoma of the breast were retrieved from the surgical pathology files

of the Tohoku University Hospital, Sendai, Japan. None of the patients had received chemotherapy, radiation therapy or hormone therapy prior to surgery. The mean age of the patients was 56.4 years (range 36–81 years). All the specimens were fixed in 10 % formalin and embedded in paraffin. The research protocol was approved by the Ethics Committee of the Tohoku University School of Medicine. 2.2 Antibodies The antibodies used were obtained from the following sources: anti-KLF15 from Atlas Antibodies (Stockholm, Sweden), antip21 from BD Biosciences (San Jose, CA, USA), ant-p27 from Merck Millipore (Hessen, Germany), anti-PARP from Cell signaling Technologies (Danvers, MA, USA), anti-β-actin from Sigma-Aldrich (St Louis, MO, USA) and anti-Topoisomerase IIα (TOP2A) from DAKO (Carpinteria, CA, USA). 2.3 Immunohistochemistry The immunohistochemical detection of the estrogen receptor (ER), the progesterone receptor (PR), HER2 and Ki-67 was performed as reported before [24]. For KLF15 detection, the Histofine Kit (Nichirei, Tokyo, Japan), based on a biotinstreptavidin methodology and a rabbit polyclonal antiKLF15 antibody, were used. After deparaffinization, antigen retrieval was performed by heating the slides in an autoclave at 120 °C for 5 min in Tris EDTA, pH 9.0 (10 mM Tris base, 1 mM EDTA). The antigen-antibody complex was visualized using 3, 3′-diaminobenzidine (DAB) solution [1 mM DAB, 50 mM Tris–HCl buffer (pH 7.6), and 0.006 % H2O2] and hematoxylin counter-staining. Vascular smooth muscle cells were used as a positive internal control [25] and adjacent ductal cells as a non-neoplastic epithelial control. For specificity verification, KLF15 immunoreactivity was abolished by peptide pre-absorption of the antibody (not shown). Since KLF15 immunoreactivity was detected in both the cytoplasm and the nucleus of the cells, the specimens examined were tentatively classified into the following four groups: no immunoreactivity in both the cytoplasm and the nucleus, immunoreactivity predominantly in the cytoplasm, immunoreactivity in both the cytoplasm and the nucleus, immunoreactivity predominantly in the nucleus [26]. ER, PR and Ki-67 immunoreactivities were all detected only in the nucleus of the carcinoma cells. The immunoreactivity of each marker was evaluated in more than 1000 carcinoma cells from each specimen, after which the immunoreactivity percentage (i.e., the labeling index, LI) was determined. Cases with an ER or a PR LI of> 10 % were considered as positive in accordance with a previous report [27]. HER2 immunoreactivity was evaluated using a grading system included in the HercepTest kit (DAKO), and strongly circumscribed HER2 membrane staining in>10 % of the carcinoma cells was considered as positive.

KLF15 in breast cancer: a novel tumor suppressor?

2.4 Breast cancer-derived cell cultures

2.7 Cell cycle analysis

Human breast cancer-derived cell lines MCF-7, T47D, MDAMB-231 and MDA-MB-453 were obtained from the American Type Culture Collection (Manassas, VA, USA). MCF-7 and T47D cells were cultured in RPMI-1640 medium (SigmaAldrich, St Louis, MO, USA) containing 10 % fetal bovine serum (FBS; Nichirei Biosciences, Tokyo, Japan) in a humidified incubator with an atmosphere containing 5 % CO2. MDAMB-231 and MDA-MB-453 cells were cultured in Leibovitz’s L-15 medium (Life Technologies, Carlsbad, CA, USA) containing 10 % FBS in a humidified incubator without CO2. Trypsin was used to detach adherent cells for passaging.

Cellular DNA contents were quantified and subjected to cell cycle analyses using flow cytometry. Briefly, cells were harvested and fixed in 70 % ethanol at −20 °C overnight. Next, the fixed cells were incubated with Propidium Iodide (PI) for 30 min at room temperature in the dark after which the DNA content of the cells was examined using a BD FACS Canto II system (BD Biosciences) at the Biomedical Research Core of the Tohoku University Graduate School of Medicine. The data were analyzed using the FlowJo software package (FlowJo, Ashland, OR, USA). 2.8 Cell preparation and immunocytochemistry

2.5 Western blotting Whole cell lysates were prepared using RIPA buffer (150 mM NaCl, 1.0 % Triton X-100, 0.5 % sodium deoxycholate, 0.1 % SDS, 50 mM Tris–HCl [pH 8.0]) supplemented with a Halt Protease Inhibitor Cocktail (Pierce Biotechnology, Rockford, IL, USA). Nuclear and cytoplasmic lysates were prepared using a CelLytic NuCLEAR Extraction Kit (Sigma-Aldrich) according to the manufacturer’s protocol. Protein samples were subjected to SDSPAGE and transferred to PVDF membranes. After blocking with TBS, containing 0.05 % Tween 20 and 5 % non-fat dry skim milk, the membranes were incubated overnight at 4 °C with primary antibodies. Next, the membranes were washed and incubated with horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit IgGs (GE Healthcare Life Sciences, Buckinghamshire, UK) for 1 h at room temperature. Immunoreactive bands were detected using ECL-Prime Western Blotting detection reagents (GE Healthcare Life Sciences) in conjunction with a Chemi Doc XRS system (Bio-Rad, Hercules, CA, USA).

2.6 KLF15 plasmid construction and transfection In order to construct an expression plasmid encoding human KLF15, a full-length cDNA fragment generated by RT-PCR was inserted into a pcDNA 3.1 (−) vector (Life Technologies). The primers used were as follows: forward, 5′-CGA ACT CGA GAC CAT GGT GGA CCA CTT ACT TC-3′ and reverse, 5′-GAC AAG CTT TCA GTT CAC GGA GCG CAC GGA-3′. The plasmid construct was sequence verified bidirectionally using a BigDye Terminator v1.1 Cycle Sequencing Kit (Life Technologies) and an Applied Biosystems 3500xL Genetic Analyzer (Life Technologies) at the Biomedical Research Unit of the Tohoku University Hospital. The construct was transfected into cells using Lipofectamine 2000 reagent (Life Technologies).

Cells transfected with the KLF15 expression plasmid or the pcDNA 3.1 (−) vector (negative control) were harvested and fixed with 4 % paraformaldehyde for 15 min at room temperature. Next, the fixed cells were washed with PBS and suspended in 0.1 % fibrinogen, after which thrombin was added to coagulate the cells. The coagulated cells were then embedded in paraffin. Immunocytochemical detection of KLF15, p21, p27 and TOP2A was performed using the biotin-streptavidin method outlined above. Antigen retrieval was achieved by heating the slides in citric acid buffer (2.0 mM citric acid and 9.0 mM trisodium citrate dehydrate [pH 6.0]) for the detection of p21, p27 and TOP2A. The immunoreactivity in each experiment was evaluated in more than 5 microscopic fields, after which the LI was determined. 2.9 Real-time PCR Total RNA was extracted using TRIzol (Life Technologies). Next, cDNA was synthesized using a QuantiTect reverse transcription kit (Qiagen, Hilden, Germany) and real-time PCR was carried out using a Light Cycler System (Roche Diagnostics, Mannheim, Germany) in conjunction with a SYBR Green PCR kit (Qiagen). The PCR primer sequences for p21, p27 and RPL13A used in this study were as follows: p21 forward 5′AAG ACC ATG TGG ACC TGT CA-3’ and reverse 5′-CGT TTG GAG TGG TAG AAA TCT G-3′; p27 forward 5′-AAG GAA GCG ACC TGC AAC-3′ and reverse 5′-CTC CAC AGA ACC GGC AT-3′ and RPL13A forward 5′-CCT GGA GGA GAA GAG GAA AG-3′ and reverse 5′-TTG AGG ACC TCT GTG TAT TT-3′. The p21 and p27 expression levels were normalized to the RPL13A expression level. 2.10 Statistical analyses All statistical analyses were performed using the JMP Pro 11.0.0 package (SAS Institute Japan, Tokyo, Japan). After immunohistochemistry, associations between KLF15 expression and clinicopathological parameters were evaluated using

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student’s t or χ2 tests. Disease free survival (DFS) and overall survival (OS) rates were analyzed using Kaplan-Meyer plots, and assessed using Wilcoxon test. For the in vitro studies, results were presented as mean±SD, and differences between two groups were evaluated by Dunnet’s test. A p value