The level of cytoskeleton remodeling proteins in the blood serum and the expression of their mRNA in the tumor tissue in metastasis of the larynx and hypopharynx cancer

Purpose: Actin-binding proteins (ABPs) and various signaling systems are involved in the metastasis of squamous cell carcinoma of the larynx and hypopharynx (SCCLH). The clinical signicance of these proteins has not yet been determined. We analyzed the relationship between the mRNA level of colin 1 (CFL1), prolin 1 (PFN1), adenylyl cyclase-associated protein 1 (CAP1), SNAIL and RND3 with metastasis in the SCCLH tissue. The serum level of the listed ABPs was estimated and the relationship of them with the expression of the corresponding mRNA was carried out. Materials and methods: The expression level of ABPs mRNA was measured by real-time RT-PCR in paired tissue samples taken from 54 patients with SCCLH (T 1-4 N 0-1 M 0 ). Expression analysis was performed using the 2 - ΔΔ CT method. The level of ABPs in the blood serum was measured by ELISA. Statistical analysis was carried out using the SPSS Statistica 20.0 software package. Results: The mRNA expression of the studied genes in tumor tissue of patients with SCCLH T 1-3 N 0 M 0 and T 2-4 N 1-2 M 0 did not differ signicantly. High expression of RND3 mRNA was accompanied by an increase in mRNA expression of all studied ABPs. In the blood serum of T 2-4 N 1-2 M 0 patients the level of PFN1 was signicantly lower by 21% and the level of CAP1 was higher by 75% compared with the group of patients with T 1-4 N 0 M 0 stage. Conclusion: According to our data RND3 is involved in the regulation of molecular cascades SCCLH metastasis. PFN1 and CAP1 serum level can be a good classier of metastases in patients with SCCLH.


Introduction
Squamous cell carcinoma of the larynx and hypopharynx (SCCLH) is characterized by rapid growth, high metastatic and invasive potential, which determines its high aggressiveness. The high mortality rate in patients with SCCLH (about 40%) in the rst year after diagnosis is a signi cant problem. A quarter of patients have clinically undetectable metastases to the lymph nodes, the manifestation of which occurs in the late postoperative period [1]. Therefore, further investigation on the mechanisms of SCCLH metastasis is necessary to improve patient outcomes.
The aggressiveness of tumorы is determined by various molecular and genetic changes, including the acquisition of migration ability by tumor cells, accompanied by reorganization of the cytoskeleton [2,3].
During metastasis tumor cells can move by the mesenchymal type, with the initiation of epithelialmesenchymal transition (EMT), and by the amoeboid type, with the activation of the mesenchymal-toamoeboid transition (MAT) [2, 3; 4; 5]. Tumor transformation of cells is characterized by EMT type 3 [2,3], accompanied by activation of transcription factors (Snail-1, Twist, Zeb, etc.), increased production of metalloproteinases, cytoskeleton reorganization by actin-binding proteins (ABPs), and other intracellular events [2,3]. Switching between EMT and MAT can be quite fast and depends on the properties of the microenvironment. The participation of proteins of the Rho family GTPases is common to all migration routes [6; 7, 8; 9]. On the cell line of mouse glioblastoma, it was shown that the induced expression of RND3 (Rho family -GTPases) inhibits the activity of Snail1, which blocks the migration of tumor cells [10]. Therefore, the study of the level and balance of mRNA of such signaling proteins as RND3 and SNAIL1 in connection with the metastatic status in patients with SCCLH is of current interest.
Various ABPs (co lin, fascin, pro lin, adenylyl cyclase-associated protein 1 (CAP-1), ezrin, etc.) are involved in the remodeling of the actin cytoskeleton, which provides the driving force of tumor cells during metastasis [4, 11, 12, 13; 15, 16]. The most important regulator of actin remodeling is the ADF (actin depolymerizing factor) / co lin family of proteins [17]. Co lin 1 (non-muscle co lin) plays an important role in the pathogenesis of various tumors [16; 17], triggers the formation of lamellipodia and ensures the direction of cell transmission [18; 19]. Co lin 1 works in tandem with such ABPs as pro lin and CAP1 [13, 20; https://www.ebi.ac.uk, https://thebiogrid.org]. Changes in the expression of CAP1, pro lin 1 and co lin 1 were noted in esophageal carcinoma [21,22]. Pro lin-1 is involved in proliferation, motility, endocytosis and other important cellular processes [23,24]. The activation of the Rho / Rock pathway leads to the phosphorylation of both co lin and pro lin [9,21,25]. Moreover, phosphorylated pro lin has a high a nity for actin and can signi cantly affect the migration activity of tumor cells [24,26]. And if the contribution of co lin to the development of a more aggressive type of tumor is unambiguous, then the participation of pro lin in tumor progression is still being studied [13,24]. CAP1 accelerates co lin-mediated disassembly of actin laments, is involved in mRNA localization and maintenance of cell polarity, motility, and receptor-mediated endocytosis [20]. The increase in CAP1 expression is associated with lymphogenous metastasis of cancer of various localizations [20,21]. We have previously shown that the development of SCCLH is associated with a change in the level of mRNA of cell motility proteins: the EMT marker of the transcription factor SNAI1 and ABPs fascin 1, ezrin, and cyclase-associated protein 1 (CAP1) [11,12]. The study of the relationship of these ABPs with the level of mRNA expression of regulatory molecules of calpains showed that SCCLH metastasis is associated with a high level of CAPN1 expression, against the background of which the level of CAP1 expression was decreased [14]. Today there is no data on the relationship of EMT-associated markers SNAI1 and RND3 with ABPs co lin1, pro lin 1, and CAP1 in the progression of SCCLH. It is also not known whether the level of these ABPs in the blood serum of SCCLH patients can re ect the progression of the disease. Therefore, the aim of the study was to estimate the relationship between the mRNA levels of EMT associated signaling molecules (SNAI1, RND3) and functional partners of ABPs (co lin1, pro lin 1, CAP1) with the processes of lymphogenous metastasis in the tissues of SCCLH. The level of co lin-1, pro lin-1, and CAP1 circulating in the blood stream was assessed in patients with SCCLH. The study will add knowledge about the mechanisms of SCCLH metastasis and will reveal potential markers for predicting the course of the disease.

Materials And Methods
The study was carried out in 54 patients with morphologically veri ed diagnosis SCCLH, aged 56.5±5.4 years. Clinical details were recorded in TABLE 1. Samples were obtained before the special antitumor treatment. Specimens of serum blood and intact epithelial tissue and primary tumor tissue were collected during videolaryngoscopy. Tissue samples were placed in an RNAlater solution (Ambion, USA) and stored at -80°C. Note: "N-" -a group of patients with SCCLH without clinically con rmed lymphogenous metastasis; "N+"a group of patients with SCCLH with clinically con rmed lymphogenous metastasis N1-2.
Evaluation of mRNA expression level.
The total mRNA pool was isolated using the CCR-50 kit (Biosilica, Russia). The quality and integrity of RNA was evaluated using capillary electrophoresis on a TapeStation device (Agilent Technologies, USA).
The synthesis of the rst cDNA strand on the RNA template was carried out using a set of reagents for reverse transcription OT-1 (Synthol, Russia). The level of gene mRNA expression was evaluated by realtime PCR (RT-qPCR) using Sybr Green technology on an iCycler ampli er (Bio-Rad, USA). To assess the nal product of the PCR reaction for the presence of primer dimers or non-speci c products, a melting curve analysis (Melt option) was used. As a reference gene, the "housekeeping" gene of the GAPDH (glyceraldehydes-3-phosphate dehydrogenase) enzyme was used. Primers were selected using the Vector NTI Advance 11.5 program and the NCBI database: ACCCATTACCTGAACTTTGACAT-3. Expression analysis was performed according to the 2 -ΔΔCT method [27]. As negative controls a reaction mixture without a matrix and with an RNA matrix without a reverse transcription step was used to control the contamination of genomic DNA.

Enzyme-linked immunosorbent assay (ELISA)
The analysis of circulating ABPs was carried out in blood serum using a Multiskan FC 100 microplate ELISA reader (ThermoFisher Scienti c), using the Human Adenylyl cyclase-associated protein 1 (CAP1) ELISA kit (Cusabio), ELISA Kit for Pro lin 1 (PFN1) ( Cloud-Clone Corp), ELISA Kit for Co lin 1 (CFL1) (Cloud-Clone Corp). Blood serum was obtained according to the approved protocol from the above patients.

Statistical analysis.
The data were processed by SPSS Statistica 20.0 software and presented as Me (Q1:Q2) and as the mean ± standard deviation. The differences were assumed to be signi cant at p<0.05. Spearman's rank correlation was performed among all molecular targets.  Table 2. The expression pro le of genes encoding proteins involved in the remodeling of the actin cytoskeleton did not show statistically signi cant differences between groups of SCCLH patients with lymphogenous metastases (T2-4N1-2M0, n=24) and SCCLH patients without clinically detectable lymphogenous metastases (T2-4N0M0, n=29). However, there was a tendency to an increase of the expression activity of the SNAI1, CFL1 and CAP1 genes in the tumor tissue of patients with SCCLH (T2-4N1-2M0).  Further the total sample of SCCLH patients was divided into groups depending on the presence of metastases in the regional lymph nodes. In the group of patients with SCCLH T1-3N0M0 the correlation analysis revealed a relationship between the mRNA levels of SNAI1-CFL1 and RND3-PFN1. In the group of patients with SCCLH T2-4N1-2M0 the SNAI1 mRNA level correlated with the CAP1 mRNA level, and RND3 correlated with CFL1 and PFN1, while the strength of the relationship between the CAP1-CFL1-PFN1 ABPs mRNA expression was stronger than in the group without metastases (Table 3). To assess the relationship between the expression level of SNAI1 and RND3 and the metastatic status of SCCLH patients the total group (N = 54, 100%) was divided depending on the level of expression of these genes by the median. The rst group consisted of patients with a high level of expression of the SNAI1 and RND3 genes ( g. 1A), the second -with a low level of expression of these genes ( g. 1B). A low expression level of SNAI1 (0 -1.09) was observed in 54% SCCLH patients of the total group 20% of which had lymphogenous metastases (N+) and 80% without metastases (N-). A high level of SNAI1 (2 -76.3) was observed in 46% SCCLH patients of the total group, among whom 47% of patients had lymphogenous metastases and 53% did not.
When analyzing the dependence of the level of ABPs mRNA expression on the level of SNAI1 expression activity signi cant differences were not found for PFN1 and CAP1 (Fig. 2C). A high level of SNAI1 expression was accompanied by a high level of mRNA expression of PFN1.
Low expression activity of RND3 (0 -1.1) was detected in tumor tissue in 44% SCCLH patients of the total group among whom the proportion of patients with lymphogenous metastases accounted for 18% with metastases and 82% without them (Fig. 1, A). High expression of RND3 mRNA (1.1-81.29) was observed in 56%, among which 51% of patients were with metastases and 49% without metastases (Fig. 1B). A high level of RND3 expression was accompanied by a high level of mRNA expression of PFN1, CFL1, and CAP1 (Fig. 2D). Moreover the dependence of PFN1 and CFL1 mRNA levels on the level of RND3expression was con rmed by correlation analysis, the results of which were described in Table 3.
1. Quantitative analysis of actin-binding proteins PFN1, CFL1 and CAP1 circulating in the blood serum of patients with squamous cell carcinoma of the larynx and hypopharynx The next stage of the study was to assess level of circulating ABPs in the blood serum of SCCLH patients by ELISA. Table 4 shows the median values of ABPs level in the blood serum of SCCLH patients in the general group (T1-4N0-2M0) and in groups depending on the presence of lymphogenous metastasis (T1-3N0M0, T2-4N1-2M0). There was a signi cant decrease in the level of pro lin-1 by 21% (p = 0.05) and an increase in the level of CAP1 by 75% (p = 0.02) in the blood serum of patients with clinically con rmed metastases to the regional lymphatic nodes compared with the group of patients without lymphogenous metastases. It should be noted that in spite of the fact that no signi cant links were revealed by correlation analysis the direction of changes in the levels of mRNA expression of PFN 1 and CAP1 in tissues was similar to the serum levels of these proteins. At the same time the expression level of CFL1 in the tumor tissue of SCCLH patients with lymphogenous metastases signi cantly increased (p = 0.08) while the protein level in the blood serum of these patients remained practically unchanged.
ROC analysis was carried out to estimate the possibility to use ABPs level for predicting SCCLH metastasis. ROC analysis is proposed as a simple and practical tool for assessing the ability of candidate predictive factors in a binary classi cation [28]. Serum PFN1 and CAP1 were the most  [29]. Thus the processes of cytoskeleton remodeling are taking place in the tumor microenvironment actively which may be re ected in changes in the total expression and level of ABPs.
Co-expression of the ABPs genes (CAP1, PFN1, and CFL1) was revealed as a result of the assessment of the presence and nature of the relationship between the levels of expression of EMT associated genes (SNAI1, RND3) and functional partners of ABPs (co lin-1, pro lin-1 and CAP1) in the tumor tissue of SCCLH patients. Changes in these mRNAs during tumor progression do not contradict the literature data [13,17,18,30]. The involvement of co lin-1 in the formation of the malignant phenotype of tumor cells is still under study. It has been shown that in vitro stimulation of metastatic cells MTLn3 by epidermal growth factor (EGF) leads to an increase in the level and activity of co lin at the anterior membrane edge, which leads to the generation of free barbed ends necessary for activation of lamellipodia formation and chemotaxis to EGF, which promotes invasion and metastasis [17,31]. It is known that CAP1 promotes disassembly of co llin-linked actin laments and acceleration of actin dynamics [17]. An increased level of CAP1 and co lin expression is associated with the invasiveness of tumor cells in cancer of some localizations [32]. Later, using structural biology methods it was shown that CAP works in tandem with co lin accelerating the depolymerization of actin laments by almost 100 times [30]. However data indicating a more complex role of CAP1 in the implementation of migration activity and invasiveness of tumor cells are accumulating. The knockdown of the CAP1 gene can lead to FAK\Rap1-mediated stimulation of cell adhesion and the formation of lamellipodia with a subsequent increase in cell motility [33]. The role of co lin and its functional partner pro lin in the aggressiveness of cancer is well covered in the literature [13]. However the dynamics and interconnection between these two proteins and their joint contribution to the formation of the metastatic phenotype of tumor cells have not been studied. The close relationship between the expression of co lin 1 and pro lin 1 in SCCLH patients is revealed and shown by us for the rst time. This data requires further more detailed study in respect that changes in the expression activity of the studied proteins mRNA are multidirectional. The functional partnership between PFN1 and CFL1 in the tumor process is described in the literature [13]. The study of mRNA co-expression of pro lin 1 and CAP1 was carried out for the rst time. However given the rather contradictory data on the role of the PFN1 and CFL1 tandem in the development of aggressiveness of various tumors [13,34,35,36] further studies of the relationship of these genes and their proteins with SCCLH progression are required.
The revealed relationship between the expression activity of RND3 and PFN1, CFL1, and CAP1 mRNA in the presented study probably indicates that a special place in molecular events during tumor metastasis belongs to RND3. It has been shown that RND3 is involved in various signaling cascades and regulation of cytoskeleton organization [8,9,10,25]. The multidirectional changes in Rnd3 expression in tumors are mentioned and associated with the mutational status [37,38]. An increased level of RND3 expression in combination with changes in the mRNA levels of CAP1, PFN1, and CFL1 and the absence of a clear association with the presence of lymphogenous metastases possibly indicates the molecular features of SCCLH progression. Further studies are required for nal conclusions about the role of Rnd3 in lymphogenous metastasis in SCCLH patients including in vitro studies which will expand the knowledge about the regulation of ABPs.
The study of pro lin 1, co lin 1 and CAP1 level in the blood serum of SCCLH patients also showed dependence on the metastatic status. The detected decrease in the level of pro lin-1 and an increase in the level of CAP1 in the blood serum in patients with clinically detectable metastases in the regional lymphatic nodes do not contradict the available literature data [13,17,18,32]. But data on the role of these ABPs in the formation of the metastatic pro le of tumor cells in SCCLH have not been described and the mechanisms of their possible functional partnership have not been disclosed. Probably not only tumor cells but also immunocompetent cells can be a source of circulating ABPs in blood serum and the results obtained may re ect the body's immune response to tumor progression [39,40].
It should be noted that the direction of changes in the expression levels of CAP1 and PFN1 mRNA in tumor tissues was similar to changes in the serum protein levelt. The revealed dependence of the serum levels of pro lin 1 and CAP1 on the presence of regional metastases indicates their participation in the pathogenesis of SCCLH. The multidirectional change in the level of these ABPs in the blood serum of SCCLH patients has been shown for the rst time.

Conclusion
Thus serum levels of pro lin 1 and CAP1 were identi ed as the most signi cant candidate markers of SCCLH metastasis on the presented group of SCCLH patients. The revealed interrelationships in the expression activity of genes associated with EMTs and ABPs genes are likely to make a certain contribution to tumor progression providing active rearrangement of the cytoskeleton. Co-expression of RND3 and mRNA of ABPs possibly provides a high migration, proliferative activity and the development of a more malignant phenotype of SCCLH. The results obtained also indicate that ABPs co lin 1, pro lin 1, and CAP1 function as partners. In general the data obtained can be used as a theoretical platform for the development of additional criteria for the prognosis of lymphogenous metastasis in SCCLH.