Overexpression of the mitochondrial ribosomal protein S18-2 in the invasive breast carcinomas
Summary. Background: Recent studies allow to consider the mitochondrial ribosomal protein S18-2 (MRPS18-2, S18-2) as a potential oncoprotein, which suggests the need for further characterization of its expression in tumors of different genesis including breast cancer (BC). The aim of the study was to analyze the expression of the S18-2 protein in BC of luminal A and basal subtypes. Materials and Methods: Operational material of BC patients stage І–ІІ (luminal A subtype, n = 30, and basal subtype, n = 10) was studied with the use of morphological, immunohistochemical, statistical and bioinformatic methods. Results: Using the immunohistochemical analysis, we found that the S18-2 protein showed the nuclear signal in 66.7% of luminal A subtype BC samples and 80.0% of basal subtype BC samples. The variability of the S18-2 expression in both the luminal A and basal subtypes of BC was revealed. Noteworthy, the number of cells expressing S18-2 in high-proliferating tumors of luminal A and basal subtype is significantly higher than in tumors with a low proliferative potential (p < 0.05). In 10 samples of luminal A subtype, the nuclear S18-2 signal was higher than median value. Moreover, the S18-2 protein was overexpressed in 4 out of such 10 samples. Metastases in the lymph nodes were found in 3 out of 4 patients with the stage II BC, low differentiation grade of the tumor and high proliferative activity. The bioinformatic analysis confirms our preliminary findings that the trend for increasing expression of the S18-2 protein in tumors correlates with the aggressiveness of malignant BC. Conclusion: The S18-2 protein may be a marker of cancer aggressiveness in BC patients.
Submitted: November 8, 2018.
*Correspondence: E-mail: email@example.com
Abbreviations used: BC — breast cancer; LI — labeling index.
Breast cancer (BC) is one of the most common cancers in the developed countries and ranks first in the structure of cancer-related morbidity and mortality among the female population of Ukraine [1, 2]. According to the National Cancer Registry, BC incidence in Ukraine is constantly increasing approaching to 63.7 per 100,000 women in 2016 .
An important feature of BC is its heterogeneity — both in terms of morphological parameters, clinical course and sensitivity to therapy, which greatly complicates the choice of a treatment protocol [3, 4].
Recent advances in BC research led to understanding that such heterogeneity results from the polymorphism of tumors at a molecular level. Determination of molecular biological characteristics of BC has allowed the development of molecular taxonomy of tumors on the basis of expression of receptors of steroid hormones (estrogens and progesterone) and also a receptor of epidermal growth factor of type 2 (HER2, or ErbB-2). The main molecular subtypes of BC were characterized, namely luminal A, luminal B, Her2-positive and basal, differing in their prognosis and response to antitumor therapy. Subsequent studies have shown the considerable heterogeneity within the framework of these subtypes, which is most pronounced in tumors of luminal A and basal subtypes [5, 6].
It should be noted that despite the significant progress made in recent years in the field of differential diagnosis and the development of targeted therapies for BC, the survival rate of patients with different BC variants remains poor due to the high incidence of recurrence. In this regard, the active search for new informative molecular markers among cell cycle regulators, oncogenes and tumor suppressor genes continues with the aim of improving the prognosis and treatment of BC patients [7, 8].
According to our recent studies, one of the indexes of the progression of hormone-dependent tumors may be the mitochondrial ribosomal protein S18-2 [9, 10].
S18-2 belongs to a family of mitochondrial ribosomal proteins involved in protein biosynthesis in mitochondria and encoded by nuclear genes. Recently, it has been found that the proteins of this family are able to perform important non-mitochondrial functions, in particular, to participate in the regulation of apoptosis and a number of cell signaling cascades .
Earlier, we have shown that the interaction of S18-2 with the RB protein releases the transcription factor E2F1, which promotes the S-phase of a cell cycle . According to our previous studies, endometrial carcinomas are characterized by elevated levels of S18-2, which is associated with a change in the expression profile of proteins associated with epithelial-mesenchymal transition . In addition, the elevated levels of the S18-2 expression were also detected in prostate carcinomas .
In MCF7 cells, overexpression of S18-2 leads to the appearance of multi-nuclear cells, indicating its possible involvement in determining the aggressiveness of breast tumors .
So, the results of experimental studies allow us to consider the S18-2 protein as a potential oncoprotein, which suggests the need for further characterization of its expression in tumors of different genesis. There are no data on S18-2 the expression in BC of various molecular subtypes and a question of its significance in the progression of a particular form of cancer remains open. The present work is aimed at studying the expression of the S18-2 protein in BC of the luminal A and basal subtypes.
MATERIALS AND METHODS
Operational material of patients with BC stage I–II of the luminal A (n = 30) or basal (n = 10) subtypes was studied. The patients did not receive special treatment before surgery. The average age of patients with tumors of luminal A subtype was 58.1 ± 2.1 and with basal — 69.3 ± 3.7 years. All patients were informed and gave a written consent to be involved in this study that was approved by the Ethical Review Board of the R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, National Academy of Sciences of Ukraine.
Morphological study was performed on hematoxylin-eosin stained tissue sections. Expression of markers was assessed by immunohistochemistry, using a polyclonal rabbit antibody against S18-2 (Proteintech group, USA) and the mouse monoclonal anti-Ki-67 antibody (clone МІВ-1) (DakoCytomation, Denmark). Antibody demasking was performed by incubating tissue sections in citrate buffer (pH 6.0), for 20 min at 96 °C in a water bath. The PolyVue detection system (Diagnostic BioSystems, USA) was used to visualize the proteins.
The results of the immunohistochemical reaction were evaluated by counting the number of stained cells, which was determined as a percent of all counted cells (labeling index, LI). Expression of markers was evaluated in 800 to 1000 cancer cells. Statistical analysis was performed, using the Statistica 8.0 software package (StatSoft, Inc.) using the nonparametric Mann — Whitney U Test and the Spearman rank correlation coefficient. Differences were considered to be significant at p < 0.05. Bioinformatic analysis of gene expression at the mRNA level was performed, using an Oncomine portal, which contains published data collected, standardized and analyzed by Compendia Bioscience (http://www.oncomine.com, April 2018, Thermo Fisher Scientific, Ann-Arbor, MI, USA).
Verification of morphological diagnosis in patients with BC has shown that BC of luminal A subtype had a structure of the lobular or, in most cases, ductal carcinoma. Neoplasms of the basal subtype were represented only by the ductal forms. The majority of tumors of the luminal A and basal subtype were of a moderate differentiation grade, i.e. 90.0% and 70.0% of samples, respectively (Table 1).
Table 1. Clinical and morphological characteristics of patients with BC
Using the S18-2 specific antibody and immunohistochemistry, we found that a nuclear S18-2 signal was observed in a large proportion of studied cases, namely in approximately 70% of luminal A subtype tumors and in 80% of samples of basal subtype (Fig. 1).
Fig. 1. Immunohistochemical detection of the S18-2 protein in breast tumors of luminal A (a, b) and basal (c, d) subtypes. Counter-stained with Mayer’s hematoxylin. Magnification × 400 (a, c) and × 1000 (b, d)
The variability in S18-2 expression and cellular localization was observed in both luminal A and basal subtypes of BC. At the same time, the variation coefficient was more pronounced in basal carcinomas (Сv = 82.0%) compared with tumors of the luminal A subtype (Сv = 68.4%).
To characterize further the pattern of S18-2 expression in each tumor, the LI was calculated. The LI of the S18-2 nuclear signal in basal carcinomas was 8.3 ± 2.4%, which was higher than in tumors of the luminal A subtype (6.9 ± 1.0%) (Table 2).
It should be noted that a significant difference (p < 0.05) was found in the number of cells with the only cytoplasmic S18-2 signal between luminal A and basal BC subtypes (LI 70.6 ± 1.7 and 35.7 ± 11.5%, respectively). This parameter was more variable in carcinomas of the basal subtype (Cv = 102.3%) compared with tumors of the luminal A subtype (Cv = 20.1%). In the further analysis, we took into account only the nuclear signal of the S18-2 protein.
Table 2. Expression of the S18-2 protein depending on the molecular subtype of BC
Note: *p < 0.05 compared with luminal A subtype.
The analysis of expression of the Ki-67 proliferation marker in BC samples has shown that the median (Me) number of Ki-67-positive cells was 17.5% for luminal A and 30.0% for basal subtypes. According to Me values, all neoplasms within subtypes were divided into subgroups with a high (Ki-67 > Me) and low (Ki-67 < Me) proliferative potential. We found that the number of cells with the strong S18-2 signal in the high-proliferative tumors of luminal A and basal subtypes was significantly higher (p < 0.05) when compared with tumors with the low proliferative potential (Fig. 2).
Calculating the Spearman rank correlation coefficient between the S18-2 expression and the proliferative activity of tumor cells, we found the positive relation in both, luminal (r = 0.3) and basal carcinomas (r = 0.56).
Based on the data on a high heterogeneity of S18-2 expression in BC, next we performed the more detailed analysis of clinical and morphological features of tumors. We found that in 66.7% of cases (in a group of patients with the luminal A subtype) the S18-2 protein showed the nuclear signal. Noteworthy, in 10 patients S18-2 expression was > Me and in four cases S18-2 was overexpressed (Fig. 3).
In 3 out of 4 BC patients with S18-2 overexpression tumors were of low differentiation grade and high proliferative activity. Moreover, metastases in the lymph nodes were detected. To corroborate our experimental data, a bioinformatic analysis of S18-2 expression at the mRNA level was performed based on the Oncomine portal, which contains the data on microarray hybridization and RNA sequencing. Importantly, expression of the MRPS18-2 gene at the mRNA level was two-fold lower in the normal breast tissue (Fig. 4, a, the left panel) and in stroma (Fig. 4, a, the right panel), compared with carcinomas, according to the data in [14, 15]. Moreover, expression of the MRPS18-2 gene progressively increased with an increase in malignancy and aggressiveness of tumors (Fig. 4, b) .
Fig. 2. Comparison of the S18-2 expression in high and low-proliferating breast tumors of luminal A and basal subtypes. Notice the higher expression of S18-2 in the high proliferating tumors
Fig. 3. Heterogeneity of S18-2 expression by levels and cellular localization in the luminal A type BC. Notice that metastases and low differentiated BC were diagnosed in patients, when the strong nuclear signal of S18-2 was observed
Fig. 4. Expression of the MRPS18-2 gene at the mRNA level, assessed with the use of the Oncomine portal. Expression of the MRPS18-2 gene was higher in BC (indicated by arrows) than in intact breast tissue (a, the left panel) or stroma (a, the right panel) [14, 15]. Noteworthy, the MRPS18-2 gene was expressed at higher levels in invasive BC (indicated by arrows) than in benign tumors and non-invasive carcinomas (b) 
As we have shown earlier, S18-2 expression increased dramatically in hormone-depending tumors, such as endometrial and prostate cancers [9, 10]. The tendency of changes in S18-2 expression of protein in invasive carcinomas as compared with non-malignant tissue, was also found earlier in endometrial cancer. According to our previous studies, there was a significant increase in the S18-2 expression in endometrial cancer compared to normal or hyperplastic endometrium. In addition, the high expression level of S18-2 protein in endometrial cancer correlated with high expression of free E2F1, which is associated with high cell proliferation and aggressiveness of malignant neoplasms . Moreover, in endometrial cancer cell lines that expressed high levels of S18-2 constitutively, the decreased E-cadherin and beta-catenin signals were registered, with simultaneous increase of vimentin .
In PC3 prostate cancer cells the epithelial-to-mesenchymal cell transition was achieved upon S18-2 overexpression, due to induction of TWIST2 and CXCR4. Importantly, cells of the PC3-subline (with the high S18-2 expression) could migrate faster both, in vitro and in vivo, that is a characteristic of metastasizing cells .
In the present paper, we found high S18-2 expression in BC, similar to cases of endometrial and prostate cancers. Also, the data of the bioinformatic analysis confirm our preliminary conclusions that the trend for increased expression of S18-2 protein in BC correlates with tumor aggressiveness.
Summarizing, we can conclude that the strong nuclear signal of the S18-2 protein in BC of luminal A and basal subtypes is associated with clinical and morphological features, characteristic for tumor progression, such as the increased proliferative activity, the low differentiation grade and also the presence of metastases in lymph nodes.
The obtained results allow us to speculate that expression levels and the nuclear localization of the S18-2 protein might serve as a marker of tumor aggressiveness in BC patients.
The study was supported by the DBPMB National Academy of Sciences of Ukraine research grant #0117U002034 “Molecular-biological factors of heterogeneity in malignant cells and variability of clinical course in patients with hormone-dependent tumors” and the Emil and Wera Cornell Foundation.
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