Impact of Silencing eEF2K Expression on the Malignant Properties of Chordoma

Impact of Silencing eEF2K Expression on the Malignant Properties of Chordoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Impact of Silencing eEF2K Expression on the Malignant Properties of Chordoma Esra Aydemir, Emre Can Tüysüz, Ömer Faruk Bayrak, Didem Tecimel, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2054247/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Jan, 2023 Read the published version in Molecular Biology Reports → Version 1 posted 4 You are reading this latest preprint version Abstract Background Eukaryotic elongation factor 2 kinase (eukaryotic elongation factor 2 kinase, eEF2K) is a calcium calmodulin dependent protein kinase that keeps the highest energy consuming cellular process of protein synthesis under check through negative regulation. EEF2K pauses global protein synthesis rates at the translational elongation step by phosphorylating its only kown substrate elongation factor 2 (eEF2), a unique translocase activity in ekaryotic cells enabling the polypeptide chain elongation. Therefore, eEF2K is thought to preserve cellular energy pools particularly upon acute development of cellular stress conditions such as nutrient deprivation, hypoxia, or infections. Recently, high expression of this enzyme has been associated with poor prognosis in an array of solid tumor types. Therefore, in a growing number of studies tremendous effort is being directed to the development of treatment methods aiming to suppress eEF2K as a novel therapeutic approach in the fight against cancer. Methods In our study, we aimed to investigate the changes in the tumorigenicity of chordoma cells in presence of gene silencing for eEF2K. Taking a transient gene silencing approach using siRNA particles, eEF2K gene expression was suppressed in chordoma cells. Results Silencing eEF2K expression was associated with a slight increase in cellular proliferation and a decrease in death rates. Furthermore, no alteration in the sensitivity of chordoma cells to chemotherapy was detected in response to the decrease in eEF2K expression which intriguingly promoted suppression of cell migratory and invasion related properties. Conclusion Our findings indicate that the loss of eEF2K expression in chordoma cell lines results in the reduction of metastatic capacity. Chordoma eEF2K migration siRNAs Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Chordoma is a slowly developing, soft tissue type of cancer that typically originates from the notochordal remnants in the embryonic period and tends to localize at the base of the skull and around the sacrum in the axial skeletal system [1]. Chordoma was first reported by Virchow in 1857 as a clivus tumor [2]. According to SEER data, the rate of diagosis reported is 1 in 10 6 in the USA, and it mostly occurs between the ages of 40–70 and its incidence under the age of 20 is 1% [3]. Although basis for gender-based predisposition is not well understood, chordoma is seen approximately twice more in women than in men, [4–7]. One of the pathophysiological features of chordomas is the overexpression of the gene called T ( BRACHYURY , mouse homolog), which is used as a biomarker for chordoma diagnosis [8]. Chordoma is highly resistant to traditional therapies like chemotherapy and relatively more responsive to radiotherapy which is -therefore- preferred as a follow up treatment option particularly ensuing radical surgeries [9–11]. Therefore, alternative treatment methods are needed because of the inadequacy of existing chemotherapy options. For that matter, understanding the molecular mechanisms driving chordoma carcinogenesis is extremely important for the discovery and development of novel targeted molecules of high efficacy. To study the molecular pathogenesis of the disease there have been several cell lines established either from primary or recurred tumors collected from patients. MUG-Chor1 and UM-Chor1 chordoma cell lines, both of which were the selected model systems for this study, are examples of such in vitro models. MUG-Chor1 originates from sacrococcygeal recurrent tumor tissue, while UM-Chor1 was established from clivial primary tumor tissue [12–14]. In an attempt to uncover a novel point of intervention for the treatment of chordoma tumors we undertook understanding the therapeutic potential of silencing of eEF2K which recently has emerged as a suitable molecular target in several solid tumors types such as breast, pancreas, lung, and brain cancer [15]. Protein synthesis is an essential cellular process for cell vialbility and is accomplished by the action of a humbling machinery comprised of several factors acting in a highly sophisticated and meticulously orchestrated fashion [16]. Furthermore, synthesis of nascent proteins dedicated to different functions to sustain cell viability claims 30–50% of cellular energy [15]. In that respect, eEF2K acts as a reversible break of this multi-member machinery to sustain metabolic energy until stress conditions are reverted back to normal [17]. EEF2K executes its break function to bring polypeptide chain elongation to a halt by phosphorylating its only known substrate, eEF2 (Eukaryotic Elongation Factor 2), which possesses a unique translocase activity in eukaryotic cells allowing the progression of ribosome on an mRNA being translated [18]. In response to acute stress, the activity of eEF2K toward eEF2 increases so that the interaction of the phosphorylated eEF2 with the ribosome is abrogated, and thereby, protein synthesis becomes paused until the inhibitory phosphates are removed to allow its re-activation and re-access to the ribosome when normalcy in the growth conditions are re-attained [19]. Majority of the cancer cells in tumor bed have a higher division capacity as well as metabolic activity compared to normal cells which forms the basis for the severity of the living conditions in their microenviroment that is deficient in oxygen, nutrients and energy and is highly acidic. Initially, the association between high eEF2K expression in cancer cells and worse degree of malignancy appeared to be contradicting the prediction that a high expression of a negative regulator for protein synthesis does not reconcile with meeting the demand for the high protein synthesis rates. However, from the perspective of cytoprotective impact of eEF2K activity through energy preservation under cellular stress conditions, it becomes explicable why cancer cells of certain tissue types favors elevation of eEF2K levels in their deprived microenvironment where they have endure. In addition, in order provide high protein synthesis rates explotation of other regulators of protein translation machinery is a frequently encountered event in cancer cells where there is a strong propensity to increase the expression of those genes important for cancer development while decreasing the expression of others that prevent cancer formation [20]. Several lines of evidence obtained in different cancer models suggest that aberrant proliferation, angiogenesis, metastasis, pro-tumorigenic immune response and cancer energetics meets such pathological needs through aberrant protein translation [16]. Regulation of eEF2K function is highly complex under the control of several mitogenic signaling pathways, including PI3K (Phosphoinositide 3-kinase), mTOR (mammalian target of rapamycin-rapamycin in mammals) and MAPK (mitogen-activated protein kinase) the activity of which blocks eEF2K activity to allow protein synthesis in accordance with proliferation inducing stimuli [21]. In contrast, AMPK, the major energy sensor signaling activates eEF2K through direct phosphorylation. In several types of cancer, de-regulation of these pathways has been shown to be pivotal events driving the oncogenesis and determining the degree of malignancy whereby more aggressive phenotypes are more frequently associated with malfunction of these pathways executing key roles in cell division, differentiation, metabolism and motility. Strikingly, depending on the nutrient and energy status of the cell, signaling through these decision making pathways for the cell fate converge on eEF2K whose outcome activity directs protein synthesis accordingly [22]. Previous studies indicate that, silencing of eEF2K protein in glioma cells is shown to trigger Tumor Necrosis Factor (TNF)-related apoptosis-inducing ligand (TRAIL) dependent death mechanism [23] and reduces the proliferation of breast cancer cells by increasing sensitivity to chemotherapeutics such as doxorubicin [24]. Likewise, knockdown of higher expression of eEF2k compared to normal in esophageal squamous cell carcinoma (ESCC) slowed down migration and proliferation rate [34]. So far there has been no such study showing the association of high expression of eEF2K and bad prognosis in chordoma and whether eEF2K has any effect on the drug resistance [34]. Based on the reports that Afatinib, an Epidermal Growth Factor Receptor (EGFR) inhibitor stops the proliferation of chordoma cells by decreasing Akt/PKB pathway (negative regulator of eEF2K activity) and lower the expression of T ( BRACHYURY , mouse homolog) [26] it is worthwhile to address whether knock down of eEFK2 in combination with conventional therapeutics would result in improved efficacy. It is important to determine the activity of eEF2K in chordoma cell lines, so that we were able to knock down the expression of eEF2K and cause a senstivity and create a higher mortality rate in chordoma cells. Materials And Methods Cell Culture Chordoma cell lines, MUG-Chor1 and UM-Chor1, were kindly gifted from the Chordoma Foundation and they were routinely checked for mycoplasma contamination and STR analysisPI. The cell lines were grown in glatin coated flasks with culture medium containing IMDM (Gibco cat no: 31980-022, ThermoScientific, USA), RPMI (11875-093, ThermoSecientific, USA), 10% FBS and 1% Penicillin-Streptomycin-Anphoteracin (PSA). Silencing of eEF2K Expression via siRNAs) Chordoma cells were transfected either with eEF2K-specific siRNA (4392420 Thermo Scientific) or negative control siRNA (AM4611 Thermo Scientific) using the liposomal-based carrier “Lipofectamine RNAi Max Transfection Reagent” (13778100, Thermo Scientific) according to the manufacturer’s instructions. Briefly, cells were grown in 6-well plates prior to the transfection and siRNAs –diluted in Opti-MEM (31985062, ThermoScientific, USA) with no serum and mixed with 3 µL Lipofectamine™ RNAiMAX- were introduced into the cells with a final concentration of 50 nM. Following a 36 hour incubation with the siRNA coctails, cells were returned to fresh medium to be harvested at 72 hour post-transfection. Gene Expression Analysis and Protein Quantification The silencing of eEF2K expression was investigated both at the mRNA and protein level on the 3 rd day following siRNA transfection. GAPDH was used as an internal control. Total RNA was isolated using Trizol reagent (15596018, ThermoScietific, USA) according to the manfacturer’s protocol. Complementary DNA was synthesized from 1000 ng RNA with the "High-Capacity cDNA Reverse Transcription Kit" (4368814, Thermo Fisher) in accordance with the manufacturer's protocol. Taqman primer probes targeting eEF2K (Hs00179434_m1,) and GAPDH (Hs02786624_g1), and Universal Taqman Master Mix (4440038, Applied Biosystem) were purchased from Thermo Fisher to run the realtime PCR according to the manufacturer’s protocol. GAPDH was used as an internal control to normalize the results. As for the analysis 2 -ΔΔCt method was used to find difference in fold change. Proteins were isolated from chordoma cell lines, which were transfected with siRNAs, by using a RIPA buffer solution (9806, CST, USA). Briefly, upon transfection, cell pellets were lysed with RIPA buffer containing protease and phosphatase inhibitor cocktail (78440, Thermo Fisher) on ice, and centrifuged for 30 minutes at 14,000 g. Protein concentration was determined by Pierce™ BCA Protein Assay Kit” (23225, Thermo Fisher, USA). Proteins were loaded onto polyacrylamide gels, transferred onto the nitrocellulose membrane, blocked with skim milk, incubated with eEEF2K (ab45168, Abcam, USA) and GAPDH (5174, Cell Signaling Technology) antibodies, and finally visualized with a chemiluminescent solution containing hydrogen peroxide in the Bio Rad imaging system. Band lengths were normalized to GAPDH by the software and protein amounts were determined. Proliferation Assay The effects of eEF2K silencing on the viability of chordoma cells were investigated on the 3rd day with a 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), viability assay (G3581, Promega, USA) which is based on the breakdown of tetrazolium salts into formazan crystals through the mitochondrial activity. Briefly, two days after the transfection, cells were seeded onto the four 96-well plates (CLS6509, Corning, USA) at a density of 5x10 3 /well and treated with 10 % MTS reagent and incubated in regular culture conditions for 1 hour in dark. Absorbance values of each well were determined by a plate reader (ELx800, Biotek Instruments, USA) at wavelength 490 nm. Results were compared with negative controls. Apoptosis Assay Early apoptotic cells were detected using the FITC Annexin V Apoptosis Detection Kit I (556547, BD Biosciences, USA) according to manufacturer’s protocol. Briefly, cells were labeled with Propium Iodide (PI) and annexin V, which is an early apoptotic marker, and the resultant intensity of luorescence was measured by using the BD Facs Calibur instrument [27]. Cell Cycle Analysis Upon inhibition of eEF2K in cells with siRNA, the cell cycle profile in cells was determined using the BD Facs Calibur device using a previously established protocol [28]. This analysis was performed 3 days after siRNA transfection. The cells were harvested and fixed in cold in 70% ethanol at least 2 hours prior to the analysis. The cells were then incubated with a solution containing 0.3 mg/ml RNAse A and 5 µg/ml PI and incubated at 37 ⁰C for 30 minutes. The cell cycle analysis was performed by measuring the fluorescence intensity of DNA using the BD Facs Calibur device. Invasion and Migration Assays Chordoma cell lines transfected with siRNA were trypsinized and seeded at 2 × 10 5 cells by dissolving in 200 µl serum-free chordoma medium into the transwells, which was then placed in wells of a-24 well plate containing 1300 µL of regular chordoma medium and kept at 37 ⁰C for 24 hours. At the end of the incubation, cells were processed for the staining protocol applied in the invasion assay (described below). For the invasion assay, cells were seeded at 2 × 10 5 cells in matrigel-coated transwells (354480, Corning, USA) according to the manufacturer's protocol. After 24 hours of incubation, the cells were fixed with 3.7% formaldehyde, permeabilized with 100% methanol, and stained with 0.1% Crystal Violet dye. The cells stained with the dye on the reverse side of the transwells were considered as the migratory and invasive cells, and compared to the unstained counterparts for the analysis. Combinatorial Treatment of eEF2K siRNA with chemotherapeutic Agents After the transfection with siRNAs, MUG-Chor1 and UM-Chor1 cells were treated for 72 hours with conventional chemo agents such as Etaposide and Cisplatin (each at 10 µM) to which they have resistance. The viability of the cells was determined by the “CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS)” according to the manufacturer's protocol described before. The combined treatment of cells silenced with eEF2K and the chemotherapeutics were compared to the negative counterparts. The percentages of the viability were determined the MTS assay. Statistical Analysis Statistical analysis was performed with GraphPad Prism 5 (GraphPad Software, La Jolla, CA) and comparisons were made using unpaired t-test and post-test, where applicable two-way analysis of variance (ANOVA) and Bonferroni's test. A P value of <0.05 was considered statistically significant. Results Transient silencing of the EEF2K in cells and the its confirmation Changes in gene and protein levels were measured upon transfection with si-eEF2K and compared to si-NC transfected counterparts. When MUG-Chor1 and UM-Chor1 cells were transiently transfected with siRNA against eEF2K, significant decreases in gene expression (Fig. 1A, B) and protein levels (Fig. 1C) were observed. Differences in EEF2K amounts are calculated by normalizing each sample to its GAPDH abundance. The effects of decrease in eEF2K levels on cell proliferation and cell cycle The diminished levels of eEF2K did not significantly affect the cellular division rate in both cell lines (Fig. 2A, B). However, silencing of eEF2K, caused a mild increase in the number of cells in S and G2/M phases in MUG-Chor1 cells (Fig. 2C). On the other hand, there was not a substantial alteration in the phases of the cell cycle in UM-Chor1 cells upon eEF2K silencing (Fig. 2D). The impacts of silencing eEF2K on cellular death Annexin V staining data indicate that silencing eEF2K did not significantly caused a change in the necrotic death and in MUG-Chor1 (Fig. 3A) and UM-Chor1 cells (Fig. 3B). The changes in the capacity of migration and invasion upon eEF2K silencing Migration and invasiveness of both cell lines were reduced upon transfection with si-eEF2K. For instance, when normalized to the control cells (no eEF2K silencing) only 60% of both MUG-Chor1 (Fig. 4A) and UM-Chor1 (Fig. 4B) cells were able to migrate through the transwells. Similar results were obtained in the invasion assay. Accordingly, around 70% of MUG-Chor1 and 50% of UM-Chor1 (Fig. 4C and 4D) cells invaded the matrigel coated transmembrane in presence of eEF2K suppression. Investigation of the change in the resistance of cells to chemotherapy with the decrease in EEF2K level Transient silencing of both cell lines with eEF2K siRNAs chemosensitized these cells to both etoposide and cis-platin partially. However, the resultant reduction in cell viability obtained via the combination treatment did not exceed 50%. MUG-Chor1 cells that are silenced for eEF2K were treated with 10 µM Cisplatin and Etoposide. Nonetheless, lower percentage of cells were viabile in groups co-treated with Cisplatin compared to those co-treated with Etoposide suggesting a more significant therapeutic outcome from Cisplatin and eEF2K siRNA dual treatment (Fig. 5A). In contrast, response of UM-Chor1 to the identical treatment scheme indicated a higher percentage of viability when compared to the MUG-Chor1 cells (Fig. 5B). All together, the combination of eEF2K silencing and chemotherapeutics caused the viability to stay above 50%; therefore, this therapy regime is not considerably detrimental on both cells. Discussion The small interference-RNAs (si-RNAs) based gene silencing has become a promising tool in developing new methods in regard to elucidating the molecular mechanisms that underlie many diseases including cancer [29]. si-RNAs are essential tools in decipfering the function of particular genes as it promotes silencing by blocking translation of mRNAs into proteins [30]. In addition to being used as a tool for understanding the gene function, in recent years siRNAs have emerged as novel drug cancdidates due to their ability to silence expression of those genes involved in oncogenesis. In our study, physiological consequences of eEF2K silencing on malignant properties of chordoma cells were investigated via transient transfection. To our knowledge, this is the first study that addresses the link between chordoma malignancy and downregulation of eEF2K expression which has been reported to exert pro-tumorigenic effects both in vitro and in vivo models of breast, ovarian, lung, glioma, meduloblastoma, hepatocellular carcinoma, pancreatic cancer and prostate cancer [31]. Suppression of eEF2K expression in the slow-growing chordoma cell line model rather hindered migratory and invasiveness in terms of malignant characteristics, while having a mior effect on the proliferative properties. EEF2K is an atypical Ca2+/calmodulin-linked protein kinase and negatively regulates mRNA translation by phosphorylating the eEF2 protein that bocks its transocase activity [32]. Various stress stimuli such as nutrient depletion, hypoxia, energy crisis, and viral infections force the cells to adapt their new environment trigger eEF2K catalytic activity toward eEF2 to turn off translation which claims more 90% of the energy dedicated to protein synthesis [33]. It is known that the levels of eEF2K is increased in various rapidly dividing tumor cells and attenuation of eEF2K expression through gene silencing methodologies potentiatiates cancer cell death induced by chemotherapeutics via various cellular death mechanisms [24]. EE2FK is overexpressed in many metastatic cancer types and has been associated with poor prognosis [17, 34]. A recent study highlighted the importance of eEF2K as a metastatic as well as prognostic biomarker in people diagnosed with gastric cancer [35]. A similar study done by Xie et al. showed that si-RNA-mediated eEF2K silencing in human lung cancer cells suppressed the tumor growth and metastasis [36]. Silencing eEF2K reduced the number of proliferating esophageal squamous cell carcinoma cells [34]. In the same study Zhu and colleagues also showed that as a result of suppression of eEF2K in cells, there was an a decrease in the number of cells in the S and M phases [37]. In contrast, our findings demonstrated that the proliferation rate of chordoma cells did not decrease upon the suppression of eEF2K which -in fact- resulted in an insignificant increase in cell division. The cell cycle profiles for both lines were found to be similar except for a slight increase in S and G2/M phases in MUG-Chor1 cells. As opposed to previous findings which suggest the inhibition of eEF2K expression in reducing the proliferation rate [38], other studies provide evidence that unleashing eEF2K activity via removal of the inhibitory upstream signaling exerts antitumorigenic effects including breast [39], intestinal, colorectal and lung cancer models [40, 41]. Particularly, translation-independent antitumorigenic effects of eEF2K attenuation detected in the non-small cell lung cancer model are interesting in the sense that an inhibitory phosphorylation of PKM2 directly by eEF2K ultimately results in suppression of glycolysis concomitant with STAT3 dependent transcription and decrease in c-Myc expression [42]. In contrast, high eEF2K expression is associated with poor patient survial and a more efficient siRNA-based silencing eEF2K in a panel of lung carcinoma cell lines resulted in a drastic suppression of colony formation, invasion and tumor volume in an earlier study [43]. Hence, it is worthwhile to understand whether differences in the gene silencing efficiency could account for the dual contrasting roles eEF2K in cancer. The role of eEF2K in protecting cells with more proliferative capacity including stem cells was demonstrated in a study done by Liao et al [44]. The authors claimed that in the absence of eEF2K expression in knock-out mice were protected against low dose ionizing radiation. In the same study, they also showed the reduction in death of stem cells of the bone marrow in the eEF2K knock out mice. The authors further explained that at lower doses of ionizing radiation (IR), the absence of eEF2K allows cells escape from (G2/M) checkpoint in the cell cycle accounting for the increased survival phenotype of the animals; however, at higher doses of IR eEF2K deficiency of the knock-out cells increases their susceptibility to undergo higher degree of mitotic catastrophe. This would explain why at least one type of cells are found arrested in S and G2/M phases of the cell cycle. Therapeutics that target eEF2K are continued to be developed and become of great interest in the treatment of triple negative breast as therapeutic effect of eEF2K knock down was demonstrated both in cancer cell lines [45] as well as in in vivo models [46]. Silencing eEF2K increases the sensitivity against lapatinib in human nasopharyngeal carcinoma cells [47], and further sensitizes human glioma cells to agents such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and temozolomide [23]. Combined therapy of an eEF2K inhibitor –mitoxantrone- and mTOR targeting increased the efficiency of treatment method against breast cancer [48]. mTOR is highly expressed in chordoma cells and is a well known biomarker [25]. Targeting mTOR is not only promising for other cancer types, but also for chordoma [49]. Peng et al. showed that targeting mTOR sensitized the cells to Cisplatin [50]. Zhang et al. showed that silencing of eEF2K increased the sensitivity of hepatocellular carcinoma cells to Cisplatin [51]. In our study, MUG-Chor1 cells responded to Cisplatin better than they did to Etoposide. The cell lines used in this study originate from different parts of the spine and therefore show varying characteristics in their response as well as drug resistance to chemotherapy. The decrease in eEF2K levels might have increased the rate of proliferation and sensitizing the cells to chemotherapy considerably. This showed us that drug resistance in chordoma cells partially attenuated when silenced with eEF2K siRNAs. As suggested by our group that there is a population of cancer stem cells in chordoma [52] and hence they might be deprived of the protecting role of eEF2K due to the silencing [44]. Therefore, the partial death observed in the chordoma cells upon treatment with chemotherapeutics in presence of silencing eEF2K could be due to losing its protective role. According to our findings, downregulation of eEF2K reduced the metastatic capacity of chordoma cells. A recent study suggests that targeting EF2K could be a promising therapeutical approach in hindering melanoma progression [53]. Other studies show that there is an association between mTOR expression and migration [54]. Considering high expression of mTOR in chordoma and the fact that eEF2K inhibiton could help potentiating the targeting mTOR pathway, we can conclude that chordoma cells may have lost their migratory capacity significantly due to the silencing of eEF2K. Angiogenesis is the process in which newly formed tumor cells, become invasive and migrate into the other parts of the body. It is found that eEF2K induces an increase in the VEGF expression and, thereby, ultimately contributes to angiogenesis. A chain of events occur upon inhibition of eEF2K including a decrease in PI3K/Akt/STAT3 pathway, which is highly expressed in chordoma [11]. This might explain the reduced migratory capacity of chordoma cell when eEF2K is silenced. Ashour et al. showed that silencing of eEF2K in pancreatic cancer cells suppressed EMT and reduced metastasis and invasion [38]. Previously we have addressed genes that regulate the invasiveness of chordoma cells and reported that TWIST -a prominent marker of Epithelial Mesenchymal Transition (EMT)- also underlies invasiveness of chordoma [55]. In a similar study, Xie et al. delineated the molecular switches that control the metastatic capabilities of breast and lung cancer cells through suppression of eEF2K suggesting EMT-modulatory roles of eEF2K loss [36]. Therefore, we investigated the impact of eEF2K silencing on the metastasis and invasion capacities of the chordoma cells in the current study. Similar to the results in the lung [31] and breast cancer study [24], our data also point out that upon eEF2K suppression, a significant reduction in metastasis and invasion capabilities both MUG-Chor1 and UM-Chor1 cells is observed. Chordoma is a rare type of bone tumor and yet classiffied as often presenting with malignant properties, including recurrence and ability for metastasis. Chordoma is also highly resistant to conventional drug-based treatment methods and therefore requires new strategies that can improves the clinical outcome. Proliferation of the cells are largely depend on the proper protein synthesis, but under suboptimal conditions, proliferation needs to be blocked until the environmental conditions go back to normal. EEF2K is an enzyme that negatively regulates the protein synthesis by phosphorylating eEF2 and causes an inhibition. When cells are exposed to energy depletion eEF2K becomes activated and trigger a reduction in overall translational rates. This mechanism is also adapted by the tumor cells as they are faced with various stress conditions including nutrient deprivation, high acidity and low oxygen levels in their poorly vascularized microenviroment, which account for these harsh living conditions as a consequence of exponential growth in the tumor mass. Chordoma cells express high eEF2K levels that might explain the slow proliferating nature of the cells. By inhibiting eEF2K we were able to force the cells to become more sensitive Cisplatin and Etoposide. Upon eEF2K depletion the chordoma cells significantly lost the metastatic and invasive capacities, which holds a promise for future preclinical studies and clinical trials. Declarations Acknowledgments : We would like to thank Fikrettin Sahin for kindly providing the chemicals and laboratory infrastructure to accomplish the study. Compliance with Ethical Standards This article does not contain any studies with human participants and/or animal participants performed by any of the authors. Author Contributions EA, DT and ET collected and analyzed data. EA and AAHD wrote the manuscript. OFB and FS reviewed and revised the manuscript. EA supervised the entire processes. All authors read and approved the final manuscript Conflict of interest/Competing interests The authors declare that they have no competing of interest Consent for publication All authors read and approved the final manuscript References Chugh R, Tawbi H, Lucas DR, et al (2007) Chordoma: the nonsarcoma primary bone tumor. Oncologist 12:1344–50. https://doi.org/10.1634/theoncologist.12-11-1344 Bell D, Raza SM, Bell AH, et al (2016) Whole-transcriptome analysis of chordoma of the skull base. Virchows Arch 469:439–449. https://doi.org/10.1007/s00428-016-1985-y Chambers KJ, Lin DT, Meier J, et al (2014) Incidence and survival patterns of cranial chordoma in the United States. Laryngoscope 124:1097–1102. https://doi.org/10.1002/lary.24420 Campbell RG, Prevedello DM, Ditzel Filho L, et al (2015) Contemporary management of clival chordomas. Curr Opin Otolaryngol Head Neck Surg 23:153–161. https://doi.org/10.1097/MOO.0000000000000140 Littman L, Reviews N (1856) Fletcher,14 Mabrey,28. 80–90 Healey JH, Lane JM (1989) Chordoma: a critical review of diagnosis and treatment. Orthop Clin North Am 20:417–426 McMaster ML, Goldstein AM, Bromley CM, et al (2001) Chordoma: incidence and survival patterns in the United States, 1973-1995. Cancer Causes Control 12:1–11. https://doi.org/10.1023/a:1008947301735 Vujovic S, Henderson S, Presneau N, et al (2006) Brachyury, a crucial regulator of notochordal development, is a novel biomarker for chordomas. J Pathol 209:157–65. https://doi.org/10.1002/path.1969 Bailey CS, Fisher CG, Boyd MC, Dvorak MFS (2006) En bloc marginal excision of a multilevel cervical chordoma. Case report. J Neurosurg Spine 4:409–414. https://doi.org/10.3171/spi.2006.4.5.409 Carrabba G, Dehdashti AR, Gentili F (2008) Surgery for clival lesions: open resection versus the expanded endoscopic endonasal approach. Neurosurg Focus 25:E7. https://doi.org/10.3171/FOC.2008.25.12.E7 Gulluoglu S, Turksoy O, Kuskucu A, et al (2016) The molecular aspects of chordoma. Neurosurg Rev 39:185–96; discussion 196. https://doi.org/10.1007/s10143-015-0663-x Brüderlein S, Sommer JB, Meltzer PS, et al (2010) Molecular characterization of putative chordoma cell lines. Sarcoma 2010:630129. https://doi.org/10.1155/2010/630129 Hsu W, Mohyeldin A, Shah SR, et al (2011) Generation of chordoma cell line JHC7 and the identification of Brachyury as a novel molecular target. J Neurosurg 115:760–9. https://doi.org/10.3171/2011.5.JNS11185 Rinner B, Froehlich EV, Buerger K, et al (2012) Establishment and detailed functional and molecular genetic characterisation of a novel sacral chordoma cell line, MUG-Chor1. Int J Oncol 40:443–451. https://doi.org/10.3892/ijo.2011.1235 Ballard DJ, Peng H-Y, Das JK, et al (2021) Insights Into the Pathologic Roles and Regulation of Eukaryotic Elongation Factor-2 Kinase. Front Mol Biosci 8:727863. https://doi.org/10.3389/fmolb.2021.727863 Bhat M, Robichaud N, Hulea L, et al (2015) Targeting the translation machinery in cancer. Nat Rev Drug Discov 14:261–278. https://doi.org/10.1038/nrd4505 Wang X, Xie J, Proud CG (2017) Eukaryotic Elongation Factor 2 Kinase (eEF2K) in Cancer. Cancers (Basel) 9:. https://doi.org/10.3390/cancers9120162 Wang X, Regufe da Mota S, Liu R, et al (2014) Eukaryotic elongation factor 2 kinase activity is controlled by multiple inputs from oncogenic signaling. Mol Cell Biol 34:4088–4103. https://doi.org/10.1128/MCB.01035-14 Leprivier G, Rotblat B, Khan D, et al (2015) Stress-mediated translational control in cancer cells. Biochim Biophys Acta 1849:845–860. https://doi.org/10.1016/j.bbagrm.2014.11.002 Silvera D, Formenti SC, Schneider RJ (2010) Translational control in cancer. Nat Rev Cancer 10:254–266. https://doi.org/10.1038/nrc2824 Roux PP, Topisirovic I (2012) Regulation of mRNA translation by signaling pathways. Cold Spring Harb Perspect Biol 4:. https://doi.org/10.1101/cshperspect.a012252 Mendoza MC, Er EE, Blenis J (2011) The Ras-ERK and PI3K-mTOR pathways: cross-talk and compensation. Trends Biochem Sci 36:320–328. https://doi.org/10.1016/j.tibs.2011.03.006 Zhang Y, Cheng Y, Zhang L, et al (2011) Inhibition of eEF-2 kinase sensitizes human glioma cells to TRAIL and down-regulates Bcl-xL expression. Biochem Biophys Res Commun 414:129–134. https://doi.org/10.1016/j.bbrc.2011.09.038 Tekedereli I, Alpay SN, Tavares CDJ, et al (2012) Targeted silencing of elongation factor 2 kinase suppresses growth and sensitizes tumors to doxorubicin in an orthotopic model of breast cancer. PLoS One 7:e41171. https://doi.org/10.1371/journal.pone.0041171 Presneau N, Shalaby A, Idowu B, et al (2009) Potential therapeutic targets for chordoma: PI3K/AKT/TSC1/TSC2/mTOR pathway. Br J Cancer 100:1406–1414. https://doi.org/10.1038/sj.bjc.6605019 Magnaghi P, Salom B, Cozzi L, et al (2018) Afatinib Is a New Therapeutic Approach in Chordoma with a Unique Ability to Target EGFR and Brachyury. Mol Cancer Ther 17:603–613. https://doi.org/10.1158/1535-7163.MCT-17-0324 Jiang S-X, Qi B, Yao W-J, et al (2017) Berberine displays antitumor activity in esophageal cancer cells in vitro. World J Gastroenterol 23:2511–2518. https://doi.org/10.3748/wjg.v23.i14.2511 Irazoqui AP, Gonzalez A, Buitrago C (2022) Effects of calcitriol on the cell cycle of rhabdomyosarcoma cells. J Steroid Biochem Mol Biol 222:106146. https://doi.org/https://doi.org/10.1016/j.jsbmb.2022.106146 Ozcan G, Ozpolat B, Coleman RL, et al (2015) Preclinical and clinical development of siRNA-based therapeutics. Adv Drug Deliv Rev 87:108–119. https://doi.org/10.1016/j.addr.2015.01.007 Hannon GJ, Rossi JJ (2004) Unlocking the potential of the human genome with RNA interference. Nature 431:371–378. https://doi.org/10.1038/nature02870 Zhang B, Zou J, Zhang Q, et al (2021) Progress in the Development of Eukaryotic Elongation Factor 2 Kinase (eEF2K) Natural Product and Synthetic Small Molecule Inhibitors for Cancer Chemotherapy. Int J Mol Sci 22:. https://doi.org/10.3390/ijms22052408 Carlberg U, Nilsson A, Nygård O (1990) Functional properties of phosphorylated elongation factor 2. Eur J Biochem 191:639–645. https://doi.org/10.1111/j.1432-1033.1990.tb19169.x Wang X, Li W, Williams M, et al (2001) Regulation of elongation factor 2 kinase by p90(RSK1) and p70 S6 kinase. EMBO J 20:4370–4379. https://doi.org/10.1093/emboj/20.16.4370 Pott LL, Hagemann S, Reis H, et al (2017) Eukaryotic elongation factor 2 is a prognostic marker and its kinase a potential therapeutic target in HCC. Oncotarget 8:11950–11962. https://doi.org/10.18632/oncotarget.14447 Jiang M, Qi L, Jin K, et al (2021) eEF2K as a novel metastatic and prognostic biomarker in gastric cancer patients. Pathol - Res Pract 225:153568. https://doi.org/https://doi.org/10.1016/j.prp.2021.153568 Xie J, Shen K, Lenchine R V, et al (2018) Eukaryotic elongation factor 2 kinase upregulates the expression of proteins implicated in cell migration and cancer cell metastasis. Int J cancer 142:1865–1877. https://doi.org/10.1002/ijc.31210 Zhu H, Song H, Chen G, et al (2017) eEF2K promotes progression and radioresistance of esophageal squamous cell carcinoma. Radiother Oncol J Eur Soc Ther Radiol Oncol 124:439–447. https://doi.org/10.1016/j.radonc.2017.04.001 Ashour AA, Abdel-Aziz A-AH, Mansour AM, et al (2014) Targeting elongation factor-2 kinase (eEF-2K) induces apoptosis in human pancreatic cancer cells. Apoptosis 19:241–258. https://doi.org/10.1007/s10495-013-0927-2 Cheng Y, Ren X, Zhang Y, et al (2013) Integrated regulation of autophagy and apoptosis by EEF2K controls cellular fate and modulates the efficacy of curcumin and velcade against tumor cells. Autophagy 9:208–219. https://doi.org/10.4161/auto.22801 Xie C-M, Liu X-Y, Sham KWY, et al (2014) Silencing of EEF2K (eukaryotic elongation factor-2 kinase) reveals AMPK-ULK1-dependent autophagy in colon cancer cells. Autophagy 10:1495–1508. https://doi.org/10.4161/auto.29164 Faller WJ, Jackson TJ, Knight JR, et al (2015) mTORC1-mediated translational elongation limits intestinal tumour initiation and growth. Nature 517:497–500. https://doi.org/10.1038/nature13896 Xiao M, Xie J, Wu Y, et al (2020) The eEF2 kinase-induced STAT3 inactivation inhibits lung cancer cell proliferation by phosphorylation of PKM2. Cell Commun Signal 18:25. https://doi.org/10.1186/s12964-020-0528-y Bircan HA, Gurbuz N, Pataer A, et al (2018) Elongation factor-2 kinase (eEF-2K) expression is associated with poor patient survival and promotes proliferation, invasion and tumor growth of lung cancer. Lung Cancer 124:31–39. https://doi.org/10.1016/j.lungcan.2018.07.027 Liao Y, Chu H-P, Hu Z, et al (2016) Paradoxical Roles of Elongation Factor-2 Kinase in Stem Cell Survival*. J Biol Chem 291:19545–19557. https://doi.org/https://doi.org/10.1074/jbc.M116.724856 Comert Onder F, Siyah P, Durdagi S, et al (2022) Novel etodolac derivatives as eukaryotic elongation factor 2 kinase (eEF2K) inhibitors for targeted cancer therapy. RSC Med Chem 13:840–849. https://doi.org/10.1039/d2md00105e Chen X, Wang K, Jiang S, et al (2022) eEF2K promotes PD-L1 stabilization through inactivating GSK3β in melanoma. J Immunother Cancer 10:. https://doi.org/10.1136/jitc-2021-004026 Liu L, Huang P, Wang Z, et al (2016) Inhibition of eEF-2 kinase sensitizes human nasopharyngeal carcinoma cells to lapatinib-induced apoptosis through the Src and Erk pathways. BMC Cancer 16:813. https://doi.org/10.1186/s12885-016-2853-5 Guan Y, Jiang S, Ye W, et al (2020) Combined treatment of mitoxantrone sensitizes breast cancer cells to rapalogs through blocking eEF-2K-mediated activation of Akt and autophagy. Cell Death Dis 11:948. https://doi.org/10.1038/s41419-020-03153-x Stacchiotti S, Marrari a, Tamborini E, et al (2009) Response to imatinib plus sirolimus in advanced chordoma. Ann Oncol 20:1886–94. https://doi.org/10.1093/annonc/mdp210 Peng D-J, Wang J, Zhou J-Y, Wu GS (2010) Role of the Akt/mTOR survival pathway in cisplatin resistance in ovarian cancer cells. Biochem Biophys Res Commun 394:600–605. https://doi.org/10.1016/j.bbrc.2010.03.029 Zhang C, Lei J-L, Zhang H, et al (2017) Calyxin Y sensitizes cisplatin-sensitive and resistant hepatocellular carcinoma cells to cisplatin through apoptotic and autophagic cell death via SCF βTrCP-mediated eEF2K degradation. Oncotarget 8:70595–70616. https://doi.org/10.18632/oncotarget.19883 Aydemir E, Bayrak OF, Sahin F, et al (2012) Characterization of cancer stem-like cells in chordoma. J Neurosurg 116:810–20. https://doi.org/10.3171/2011.12.JNS11430 Deng G, Zeng F, He Y, et al (2022) EEF2K silencing inhibits tumour progression through repressing SPP1 and synergises with BET inhibitors in melanoma. Clin Transl Med 12:e722. https://doi.org/10.1002/ctm2.722 Zhou H, Huang S (2011) Role of mTOR signaling in tumor cell motility, invasion and metastasis. Curr Protein Pept Sci 12:30–42. https://doi.org/10.2174/138920311795659407 Aydemir E, Kaşikci E, Coşkunçelebi B, et al (2018) The effect of TWIST silencing in metastatic chordoma cells. Turkish J Biol 42:. https://doi.org/10.3906/biy-1801-17 Cite Share Download PDF Status: Published Journal Publication published 18 Jan, 2023 Read the published version in Molecular Biology Reports → Version 1 posted Reviewers agreed at journal 06 Oct, 2022 Reviewers invited by journal 16 Sep, 2022 Editor assigned by journal 13 Sep, 2022 First submitted to journal 12 Sep, 2022 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2054247","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":137186569,"identity":"c5356fdb-528c-4f99-969a-9aec21fd438a","order_by":0,"name":"Esra Aydemir","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6ElEQVRIiWNgGAWjYJACCRDil2BsgLAZEojUIjkDWcsBwloYGAxuwPkEtPCznzG88THHIt/4dnPbZ94dFkCRHAPmj3twa5HsyTG2nLlNwnLbnYPNs3nPAF3Y88aA4cAz3FoMDuSYSfNukzAwu5HYzMzbJgF0YQ5QCx6X2Z9/Yyb9F6jFeAZUiz0hLQYSQFsYgVoMJGC2SBDQInHjWbFlL1CLBNAvjHPbJHgkzjwrOHAGjxb+/uSNN35uqzPgn93+mOFtW50cf3vyxgcVeLQwMHAYoHB5QAReDQwM7A/wy4+CUTAKRsEoAADuZ07H6eUXRQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-6965-2838","institution":"Biruni Universitesi","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Esra","middleName":"","lastName":"Aydemir","suffix":""},{"id":137186570,"identity":"484931c2-4253-4517-b071-5c9b1db68bbf","order_by":1,"name":"Emre Can Tüysüz","email":"","orcid":"","institution":"Lund University: Lunds Universitet","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Emre","middleName":"Can","lastName":"Tüysüz","suffix":""},{"id":137186571,"identity":"aeb73c68-79b3-404d-92d1-d1d649462c2c","order_by":2,"name":"Ömer Faruk Bayrak","email":"","orcid":"","institution":"Yeditepe Üniversitesi Tıp Fakültesi: Yeditepe Universitesi Tip Fakultesi","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ömer","middleName":"Faruk","lastName":"Bayrak","suffix":""},{"id":137186572,"identity":"1fea4f5c-718b-4940-a285-74490ba9f27e","order_by":3,"name":"Didem Tecimel","email":"","orcid":"","institution":"Yeditepe University: Yeditepe Universitesi","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Didem","middleName":"","lastName":"Tecimel","suffix":""},{"id":137186573,"identity":"f230f6c3-ffda-4d9f-9987-b721c4dee053","order_by":4,"name":"Ayşen Aslı Hızlı-Deniz","email":"","orcid":"","institution":"Yeditepe University: Yeditepe Universitesi","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ayşen","middleName":"Aslı","lastName":"Hızlı-Deniz","suffix":""},{"id":137186574,"identity":"51d74efe-4bdb-441d-a8a1-d7f2fe9d4bbb","order_by":5,"name":"Fikrettin Şahin","email":"","orcid":"","institution":"Yeditepe University: Yeditepe Universitesi","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Fikrettin","middleName":"","lastName":"Şahin","suffix":""}],"badges":[],"createdAt":"2022-09-11 16:45:01","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2054247/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2054247/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11033-023-08257-z","type":"published","date":"2023-01-18T18:24:19+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":26671710,"identity":"beadd05c-e74f-4482-863d-306eb892780f","added_by":"auto","created_at":"2022-09-19 20:33:21","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":97176,"visible":true,"origin":"","legend":"\u003cp\u003eThe confirmation of knock-down by gene expression and western blot analyses\u003c/p\u003e","description":"","filename":"MBRFigure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2054247/v1/f90ae82707f043868137d5b2.jpg"},{"id":26671711,"identity":"e950ef47-4e69-4d07-ac7c-17287739b732","added_by":"auto","created_at":"2022-09-19 20:33:21","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3757597,"visible":true,"origin":"","legend":"\u003cp\u003eThe changes in the proliferation status of chordoma cells upon silencing. \u003cstrong\u003eA\u003c/strong\u003e MUG-Chor1 cells \u003cstrong\u003eB \u003c/strong\u003eUM-Chor1 cells. The changes in the cell cycle profile of chordoma cells upon silencing. \u003cstrong\u003eC\u003c/strong\u003e MUG-Chor1 cells and \u003cstrong\u003eD\u003c/strong\u003e UM-Chor1 cells\u003c/p\u003e","description":"","filename":"MBRfigure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2054247/v1/0165c6221756f1f5aacc28e3.jpg"},{"id":26671707,"identity":"fb33b0b6-2a79-4a3e-ba95-4e759624dc6d","added_by":"auto","created_at":"2022-09-19 20:33:20","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":462891,"visible":true,"origin":"","legend":"\u003cp\u003eThe cell death profile of cells via Annexin V analysis after the silencing with si-eEF2K. \u003cstrong\u003eA\u003c/strong\u003eMUG-Chor1 cells \u003cstrong\u003eB \u003c/strong\u003eUM-Chor1 cells\u003c/p\u003e","description":"","filename":"MBRFigure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2054247/v1/778f32dcf17f5e565c73f8cf.jpg"},{"id":26672345,"identity":"30c45093-9107-4144-9a07-097e3516400b","added_by":"auto","created_at":"2022-09-19 20:38:20","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":761310,"visible":true,"origin":"","legend":"\u003cp\u003eThe numbers of migratory cells upon si-eEF2K silencing on chordoma cells \u003cstrong\u003eA\u003c/strong\u003e MUG-Chor1 cells \u003cstrong\u003eB \u003c/strong\u003eUM-Chor1 cells. The number of invasive cells upon si-eEF2K silencing on chordoma cells \u003cstrong\u003eC\u003c/strong\u003e MUG-Chor1 cells and \u003cstrong\u003eD\u003c/strong\u003eUM-Chor1 cells\u003c/p\u003e","description":"","filename":"MBRFigure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2054247/v1/48b27d594240fe29ef20f3b4.jpg"},{"id":26671709,"identity":"1487de2e-5696-476b-b6d8-c453cde73024","added_by":"auto","created_at":"2022-09-19 20:33:20","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":614376,"visible":true,"origin":"","legend":"\u003cp\u003eThe chemosensitivity assay shows the number of viable cells synergistically upon silencing with si-eEF2K and drugs (Etoposide and Cisplatin). \u003cstrong\u003eA\u003c/strong\u003e MUG-Chor1 cells \u003cstrong\u003eB \u003c/strong\u003eUM-Chor1 cells\u003c/p\u003e","description":"","filename":"MBRFigure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2054247/v1/647ed8e7cd7a284798ceb4ae.jpg"},{"id":44717716,"identity":"406a7e90-ecf5-4454-b385-5b37b452b9f3","added_by":"auto","created_at":"2023-10-16 18:39:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":794874,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2054247/v1/218fa835-591f-40f5-9480-4caca60c06ac.pdf"}],"financialInterests":"","formattedTitle":"Impact of Silencing eEF2K Expression on the Malignant Properties of Chordoma","fulltext":[{"header":"Introduction","content":"\u003cp\u003eChordoma is a slowly developing, soft tissue type of cancer that typically originates from the notochordal remnants in the embryonic period and tends to localize at the base of the skull and around the sacrum in the axial skeletal system [1]. Chordoma was first reported by Virchow in 1857 as a clivus tumor [2]. According to SEER data, the rate of diagosis reported is 1 in 10\u003csup\u003e6\u003c/sup\u003e in the USA, and it mostly occurs between the ages of 40\u0026ndash;70 and its incidence under the age of 20 is 1% [3]. Although basis for gender-based predisposition is not well understood, chordoma is seen approximately twice more in women than in men, [4\u0026ndash;7]. One of the pathophysiological features of chordomas is the overexpression of the gene called \u003cem\u003eT\u003c/em\u003e (\u003cem\u003eBRACHYURY\u003c/em\u003e, mouse homolog), which is used as a biomarker for chordoma diagnosis [8]. Chordoma is highly resistant to traditional therapies like chemotherapy and relatively more responsive to radiotherapy which is -therefore- preferred as a follow up treatment option particularly ensuing radical surgeries [9\u0026ndash;11]. Therefore, alternative treatment methods are needed because of the inadequacy of existing chemotherapy options. For that matter, understanding the molecular mechanisms driving chordoma carcinogenesis is extremely important for the discovery and development of novel targeted molecules of high efficacy. To study the molecular pathogenesis of the disease there have been several cell lines established either from primary or recurred tumors collected from patients. MUG-Chor1 and UM-Chor1 chordoma cell lines, both of which were the selected model systems for this study, are examples of such in vitro models. MUG-Chor1 originates from sacrococcygeal recurrent tumor tissue, while UM-Chor1 was established from clivial primary tumor tissue [12\u0026ndash;14].\u003c/p\u003e \u003cp\u003eIn an attempt to uncover a novel point of intervention for the treatment of chordoma tumors we undertook understanding the therapeutic potential of silencing of eEF2K which recently has emerged as a suitable molecular target in several solid tumors types such as breast, pancreas, lung, and brain cancer [15].\u003c/p\u003e \u003cp\u003eProtein synthesis is an essential cellular process for cell vialbility and is accomplished by the action of a humbling machinery comprised of several factors acting in a highly sophisticated and meticulously orchestrated fashion [16]. Furthermore, synthesis of nascent proteins dedicated to different functions to sustain cell viability claims 30\u0026ndash;50% of cellular energy [15]. In that respect, eEF2K acts as a reversible break of this multi-member machinery to sustain metabolic energy until stress conditions are reverted back to normal [17]. EEF2K executes its break function to bring polypeptide chain elongation to a halt by phosphorylating its only known substrate, eEF2 (Eukaryotic Elongation Factor 2), which possesses a unique translocase activity in eukaryotic cells allowing the progression of ribosome on an mRNA being translated [18]. In response to acute stress, the activity of eEF2K toward eEF2 increases so that the interaction of the phosphorylated eEF2 with the ribosome is abrogated, and thereby, protein synthesis becomes paused until the inhibitory phosphates are removed to allow its re-activation and re-access to the ribosome when normalcy in the growth conditions are re-attained [19].\u003c/p\u003e \u003cp\u003eMajority of the cancer cells in tumor bed have a higher division capacity as well as metabolic activity compared to normal cells which forms the basis for the severity of the living conditions in their microenviroment that is deficient in oxygen, nutrients and energy and is highly acidic. Initially, the association between high eEF2K expression in cancer cells and worse degree of malignancy appeared to be contradicting the prediction that a high expression of a negative regulator for protein synthesis does not reconcile with meeting the demand for the high protein synthesis rates. However, from the perspective of cytoprotective impact of eEF2K activity through energy preservation under cellular stress conditions, it becomes explicable why cancer cells of certain tissue types favors elevation of eEF2K levels in their deprived microenvironment where they have endure. In addition, in order provide high protein synthesis rates explotation of other regulators of protein translation machinery is a frequently encountered event in cancer cells where there is a strong propensity to increase the expression of those genes important for cancer development while decreasing the expression of others that prevent cancer formation [20]. Several lines of evidence obtained in different cancer models suggest that aberrant proliferation, angiogenesis, metastasis, pro-tumorigenic immune response and cancer energetics meets such pathological needs through aberrant protein translation [16].\u003c/p\u003e \u003cp\u003eRegulation of eEF2K function is highly complex under the control of several mitogenic signaling pathways, including PI3K (Phosphoinositide 3-kinase), mTOR (mammalian target of rapamycin-rapamycin in mammals) and MAPK (mitogen-activated protein kinase) the activity of which blocks eEF2K activity to allow protein synthesis in accordance with proliferation inducing stimuli [21]. In contrast, AMPK, the major energy sensor signaling activates eEF2K through direct phosphorylation. In several types of cancer, de-regulation of these pathways has been shown to be pivotal events driving the oncogenesis and determining the degree of malignancy whereby more aggressive phenotypes are more frequently associated with malfunction of these pathways executing key roles in cell division, differentiation, metabolism and motility. Strikingly, depending on the nutrient and energy status of the cell, signaling through these decision making pathways for the cell fate converge on eEF2K whose outcome activity directs protein synthesis accordingly [22].\u003c/p\u003e \u003cp\u003ePrevious studies indicate that, silencing of eEF2K protein in glioma cells is shown to trigger Tumor Necrosis Factor (TNF)-related apoptosis-inducing ligand (TRAIL) dependent death mechanism [23] and reduces the proliferation of breast cancer cells by increasing sensitivity to chemotherapeutics such as doxorubicin [24]. Likewise, knockdown of higher expression of eEF2k compared to normal in esophageal squamous cell carcinoma (ESCC) slowed down migration and proliferation rate [34]. So far there has been no such study showing the association of high expression of eEF2K and bad prognosis in chordoma and whether eEF2K has any effect on the drug resistance [34].\u003c/p\u003e \u003cp\u003eBased on the reports that Afatinib, an Epidermal Growth Factor Receptor (EGFR) inhibitor stops the proliferation of chordoma cells by decreasing Akt/PKB pathway (negative regulator of eEF2K activity) and lower the expression of \u003cem\u003eT\u003c/em\u003e (\u003cem\u003eBRACHYURY\u003c/em\u003e, mouse homolog) [26] it is worthwhile to address whether knock down of eEFK2 in combination with conventional therapeutics would result in improved efficacy. It is important to determine the activity of eEF2K in chordoma cell lines, so that we were able to knock down the expression of eEF2K and cause a senstivity and create a higher mortality rate in chordoma cells.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003eCell Culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChordoma cell lines, MUG-Chor1 and UM-Chor1, were kindly gifted from the Chordoma Foundation and they were routinely checked for mycoplasma contamination and STR analysisPI. The cell lines were grown in glatin coated flasks with culture medium containing IMDM (Gibco cat no: 31980-022, ThermoScientific, USA), RPMI (11875-093, ThermoSecientific, USA), 10% FBS and 1% Penicillin-Streptomycin-Anphoteracin (PSA).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSilencing of eEF2K Expression via siRNAs)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChordoma cells were transfected either with eEF2K-specific siRNA (4392420 Thermo Scientific) or negative control siRNA (AM4611 Thermo Scientific) using the liposomal-based carrier \u0026ldquo;Lipofectamine RNAi Max Transfection Reagent\u0026rdquo; (13778100, Thermo Scientific) according to the manufacturer\u0026rsquo;s instructions. Briefly, cells were grown in 6-well plates prior to the transfection and siRNAs \u0026ndash;diluted in Opti-MEM (31985062, ThermoScientific, USA) with no serum and mixed with 3 \u0026micro;L Lipofectamine\u0026trade; RNAiMAX- were introduced into the cells with a final concentration of 50 nM. Following a 36 hour incubation with the siRNA coctails, cells were returned to fresh medium to be harvested at 72 hour post-transfection.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGene Expression Analysis and Protein Quantification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe silencing of eEF2K expression was investigated both at the mRNA and protein level on the 3\u003csup\u003erd\u003c/sup\u003e day following siRNA transfection. GAPDH was used as an internal control. Total RNA was isolated using Trizol reagent (15596018, ThermoScietific, USA) according to the manfacturer\u0026rsquo;s protocol. Complementary DNA was synthesized from 1000 ng RNA with the \u0026quot;High-Capacity cDNA Reverse Transcription Kit\u0026quot; (4368814, Thermo Fisher) in accordance with the manufacturer\u0026apos;s protocol. Taqman primer probes targeting eEF2K (Hs00179434_m1,) and GAPDH (Hs02786624_g1), and Universal Taqman Master Mix (4440038, Applied Biosystem) were purchased from Thermo Fisher to run the realtime PCR according to the manufacturer\u0026rsquo;s protocol. GAPDH was used as an internal control to normalize the results. As for the analysis 2\u003csup\u003e-\u0026Delta;\u0026Delta;Ct\u003c/sup\u003e method was used to find difference in fold change.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eProteins were isolated from chordoma cell lines, which were transfected with siRNAs, by using a RIPA buffer solution (9806, CST, USA). Briefly, upon transfection, cell pellets were lysed with RIPA buffer containing protease and phosphatase inhibitor cocktail (78440, Thermo Fisher) on ice, and centrifuged for 30 minutes at 14,000 g. Protein concentration was determined by Pierce\u0026trade; BCA Protein Assay Kit\u0026rdquo; (23225, Thermo Fisher, USA). Proteins were loaded onto polyacrylamide gels, transferred onto the nitrocellulose membrane, blocked with skim milk, incubated with eEEF2K (ab45168, Abcam, USA) and GAPDH (5174, Cell Signaling Technology) antibodies, and finally visualized with a chemiluminescent solution containing hydrogen peroxide in the Bio Rad imaging system. Band lengths were normalized to GAPDH by the software and protein amounts were determined.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProliferation Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe effects of eEF2K silencing on the viability of chordoma cells were investigated on the 3rd day with a 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), viability assay (G3581, Promega, USA) which is based on the breakdown of tetrazolium salts into formazan crystals through the mitochondrial activity. Briefly, two days after the transfection, cells were seeded onto the four 96-well plates (CLS6509, Corning, USA) at a density of 5x10\u003csup\u003e3\u003c/sup\u003e/well and treated with 10 % MTS reagent and incubated in regular culture conditions for 1 hour in dark. Absorbance values of each well were determined by a plate reader (ELx800, Biotek Instruments, USA) at wavelength 490 nm. Results were compared with negative controls.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eApoptosis Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEarly apoptotic cells were detected using the FITC Annexin V Apoptosis Detection Kit I (556547, BD Biosciences, USA) according to manufacturer\u0026rsquo;s protocol. Briefly, cells were labeled with Propium Iodide (PI) and annexin V, which is an early apoptotic marker, and the resultant intensity of luorescence was measured by using the BD Facs Calibur instrument\u0026nbsp;[27].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell Cycle Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUpon inhibition of eEF2K in cells with siRNA, the cell cycle profile in cells was determined using the BD Facs Calibur device using a previously established protocol\u0026nbsp;[28]. This analysis was performed 3 days after siRNA transfection. The cells were harvested and fixed in cold in 70% ethanol at least 2 hours prior to the analysis. The cells were then incubated with a solution containing 0.3 mg/ml RNAse A and 5 \u0026micro;g/ml PI and incubated at 37 ⁰C for 30 minutes. The cell cycle analysis was performed by measuring the fluorescence intensity of DNA using the BD Facs Calibur device.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInvasion and Migration Assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChordoma cell lines transfected with siRNA were trypsinized and seeded at 2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells by dissolving in 200 \u0026micro;l serum-free chordoma medium into the transwells, which was then placed in wells of a-24 well plate containing 1300 \u0026micro;L of regular chordoma medium and kept at 37 ⁰C for 24 hours. At the end of the incubation, cells were processed for the staining protocol applied in the invasion assay (described below). For the invasion assay, cells were seeded at 2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells in matrigel-coated transwells (354480, Corning, USA) according to the manufacturer\u0026apos;s protocol. After 24 hours of incubation, the cells were fixed with 3.7% formaldehyde, permeabilized with 100% methanol, and stained with 0.1% Crystal Violet dye. The cells stained with the dye on the reverse side of the transwells were considered as the migratory and invasive cells, and compared to the unstained counterparts for the analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCombinatorial Treatment of eEF2K siRNA with chemotherapeutic Agents\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter the transfection with siRNAs, MUG-Chor1 and UM-Chor1 cells were treated for 72 hours with conventional chemo agents such as Etaposide and Cisplatin (each at 10 \u0026micro;M) to which they have resistance. The viability of the cells was determined by the \u0026ldquo;CellTiter 96\u0026reg; AQueous One Solution Cell Proliferation Assay (MTS)\u0026rdquo; according to the manufacturer\u0026apos;s protocol described before. The combined treatment of cells silenced with eEF2K and the chemotherapeutics were compared to the negative counterparts. The percentages of the viability were determined the MTS assay. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analysis was performed with GraphPad Prism 5 (GraphPad Software, La Jolla, CA) and comparisons were made using unpaired t-test and post-test, where applicable two-way analysis of variance (ANOVA) and Bonferroni\u0026apos;s test. A P value of \u0026lt;0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eTransient silencing of the \u003cem\u003eEEF2K\u003c/em\u003e in cells and the its confirmation\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChanges in gene and protein levels were measured upon transfection with si-eEF2K and compared to si-NC transfected counterparts. When MUG-Chor1 and UM-Chor1 cells were transiently transfected with siRNA against eEF2K, significant decreases in gene expression (Fig. 1A, B) and protein levels (Fig. 1C) were observed. Differences in EEF2K amounts are calculated by normalizing each sample to its GAPDH abundance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe effects of decrease in eEF2K levels on cell proliferation and cell cycle\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe diminished levels of eEF2K did not significantly affect the cellular division rate in both cell lines (Fig. 2A, B). However, silencing of eEF2K, caused a mild increase in the number of cells in S and G2/M phases in MUG-Chor1 cells (Fig. 2C). On the other hand, there was not a substantial alteration in the phases of the cell cycle in UM-Chor1 cells upon eEF2K silencing (Fig. 2D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe impacts of silencing eEF2K on cellular death\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnnexin V staining data indicate that silencing eEF2K did not significantly caused a change in the necrotic death and in MUG-Chor1 (Fig. 3A) and UM-Chor1 cells (Fig. 3B).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe changes in the capacity of migration and invasion upon eEF2K silencing\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMigration and invasiveness of both cell lines were reduced upon transfection with si-eEF2K. For instance, when normalized to the control cells (no eEF2K silencing) only 60% of both MUG-Chor1 (Fig. 4A) and UM-Chor1 (Fig. 4B) cells were able to migrate through the transwells. Similar results were obtained in the invasion assay. Accordingly, around 70% of MUG-Chor1 and 50% of UM-Chor1 (Fig. 4C and 4D) cells invaded the matrigel coated transmembrane in presence of eEF2K suppression.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInvestigation of the change in the resistance of cells to chemotherapy with the decrease in EEF2K level\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTransient silencing of both cell lines with eEF2K siRNAs chemosensitized these cells to both etoposide and cis-platin partially. However, the resultant reduction in cell viability obtained via the combination treatment did not exceed 50%. MUG-Chor1 cells that are silenced for eEF2K were treated with 10 \u0026micro;M Cisplatin and Etoposide. Nonetheless, lower percentage of cells were viabile in groups co-treated with Cisplatin compared to those co-treated with Etoposide suggesting a more significant therapeutic outcome from Cisplatin and eEF2K siRNA dual treatment (Fig. 5A). In contrast, response of UM-Chor1 to the identical treatment scheme indicated a higher percentage of viability when compared to the MUG-Chor1 cells (Fig. 5B). All together, the combination of eEF2K silencing and chemotherapeutics caused the viability to stay above 50%; therefore, this therapy regime is not considerably detrimental on both cells.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe small interference-RNAs (si-RNAs) based gene silencing has become a promising tool in developing new methods in regard to elucidating the molecular mechanisms that underlie many diseases including cancer [29]. si-RNAs are essential tools in decipfering the function of particular genes as it promotes silencing by blocking translation of mRNAs into proteins [30]. In addition to being used as a tool for understanding the gene function, in recent years siRNAs have emerged as novel drug cancdidates due to their ability to silence expression of those genes involved in oncogenesis. In our study, physiological consequences of eEF2K silencing on malignant properties of chordoma cells were investigated via transient transfection. To our knowledge, this is the first study that addresses the link between chordoma malignancy and downregulation of eEF2K expression which has been reported to exert pro-tumorigenic effects both in vitro and in vivo models of breast, ovarian, lung, glioma, meduloblastoma, hepatocellular carcinoma, pancreatic cancer and prostate cancer [31]. Suppression of eEF2K expression in the slow-growing chordoma cell line model rather hindered migratory and invasiveness in terms of malignant characteristics, while having a mior effect on the proliferative properties.\u003c/p\u003e \u003cp\u003eEEF2K is an atypical Ca2+/calmodulin-linked protein kinase and negatively regulates mRNA translation by phosphorylating the eEF2 protein that bocks its transocase activity [32]. Various stress stimuli such as nutrient depletion, hypoxia, energy crisis, and viral infections force the cells to adapt their new environment trigger eEF2K catalytic activity toward eEF2 to turn off translation which claims more 90% of the energy dedicated to protein synthesis [33]. It is known that the levels of eEF2K is increased in various rapidly dividing tumor cells and attenuation of eEF2K expression through gene silencing methodologies potentiatiates cancer cell death induced by chemotherapeutics via various cellular death mechanisms [24]. EE2FK is overexpressed in many metastatic cancer types and has been associated with poor prognosis [17, 34]. A recent study highlighted the importance of eEF2K as a metastatic as well as prognostic biomarker in people diagnosed with gastric cancer [35]. A similar study done by Xie et al. showed that si-RNA-mediated eEF2K silencing in human lung cancer cells suppressed the tumor growth and metastasis [36]. Silencing eEF2K reduced the number of proliferating esophageal squamous cell carcinoma cells [34]. In the same study Zhu and colleagues also showed that as a result of suppression of eEF2K in cells, there was an a decrease in the number of cells in the S and M phases [37]. In contrast, our findings demonstrated that the proliferation rate of chordoma cells did not decrease upon the suppression of eEF2K which -in fact- resulted in an insignificant increase in cell division. The cell cycle profiles for both lines were found to be similar except for a slight increase in S and G2/M phases in MUG-Chor1 cells.\u003c/p\u003e \u003cp\u003eAs opposed to previous findings which suggest the inhibition of eEF2K expression in reducing the proliferation rate [38], other studies provide evidence that unleashing eEF2K activity via removal of the inhibitory upstream signaling exerts antitumorigenic effects including breast [39], intestinal, colorectal and lung cancer models [40, 41]. Particularly, translation-independent antitumorigenic effects of eEF2K attenuation detected in the non-small cell lung cancer model are interesting in the sense that an inhibitory phosphorylation of PKM2 directly by eEF2K ultimately results in suppression of glycolysis concomitant with STAT3 dependent transcription and decrease in c-Myc expression [42]. In contrast, high eEF2K expression is associated with poor patient survial and a more efficient siRNA-based silencing eEF2K in a panel of lung carcinoma cell lines resulted in a drastic suppression of colony formation, invasion and tumor volume in an earlier study [43]. Hence, it is worthwhile to understand whether differences in the gene silencing efficiency could account for the dual contrasting roles eEF2K in cancer.\u003c/p\u003e \u003cp\u003eThe role of eEF2K in protecting cells with more proliferative capacity including stem cells was demonstrated in a study done by Liao et al [44]. The authors claimed that in the absence of eEF2K expression in knock-out mice were protected against low dose ionizing radiation. In the same study, they also showed the reduction in death of stem cells of the bone marrow in the eEF2K knock out mice. The authors further explained that at lower doses of ionizing radiation (IR), the absence of eEF2K allows cells escape from (G2/M) checkpoint in the cell cycle accounting for the increased survival phenotype of the animals; however, at higher doses of IR eEF2K deficiency of the knock-out cells increases their susceptibility to undergo higher degree of mitotic catastrophe. This would explain why at least one type of cells are found arrested in S and G2/M phases of the cell cycle.\u003c/p\u003e \u003cp\u003eTherapeutics that target eEF2K are continued to be developed and become of great interest in the treatment of triple negative breast as therapeutic effect of eEF2K knock down was demonstrated both in cancer cell lines [45] as well as in \u003cem\u003ein vivo\u003c/em\u003e models [46]. Silencing eEF2K increases the sensitivity against lapatinib in human nasopharyngeal carcinoma cells [47], and further sensitizes human glioma cells to agents such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and temozolomide [23]. Combined therapy of an eEF2K inhibitor \u0026ndash;mitoxantrone- and mTOR targeting increased the efficiency of treatment method against breast cancer [48]. mTOR is highly expressed in chordoma cells and is a well known biomarker [25]. Targeting mTOR is not only promising for other cancer types, but also for chordoma [49]. Peng et al. showed that targeting mTOR sensitized the cells to Cisplatin [50]. Zhang et al. showed that silencing of eEF2K increased the sensitivity of hepatocellular carcinoma cells to Cisplatin [51]. In our study, MUG-Chor1 cells responded to Cisplatin better than they did to Etoposide. The cell lines used in this study originate from different parts of the spine and therefore show varying characteristics in their response as well as drug resistance to chemotherapy. The decrease in eEF2K levels might have increased the rate of proliferation and sensitizing the cells to chemotherapy considerably. This showed us that drug resistance in chordoma cells partially attenuated when silenced with eEF2K siRNAs. As suggested by our group that there is a population of cancer stem cells in chordoma [52] and hence they might be deprived of the protecting role of eEF2K due to the silencing [44]. Therefore, the partial death observed in the chordoma cells upon treatment with chemotherapeutics in presence of silencing eEF2K could be due to losing its protective role.\u003c/p\u003e \u003cp\u003eAccording to our findings, downregulation of eEF2K reduced the metastatic capacity of chordoma cells. A recent study suggests that targeting EF2K could be a promising therapeutical approach in hindering melanoma progression [53].\u003c/p\u003e \u003cp\u003eOther studies show that there is an association between mTOR expression and migration [54]. Considering high expression of mTOR in chordoma and the fact that eEF2K inhibiton could help potentiating the targeting mTOR pathway, we can conclude that chordoma cells may have lost their migratory capacity significantly due to the silencing of eEF2K.\u003c/p\u003e \u003cp\u003eAngiogenesis is the process in which newly formed tumor cells, become invasive and migrate into the other parts of the body. It is found that eEF2K induces an increase in the VEGF expression and, thereby, ultimately contributes to angiogenesis. A chain of events occur upon inhibition of eEF2K including a decrease in PI3K/Akt/STAT3 pathway, which is highly expressed in chordoma [11]. This might explain the reduced migratory capacity of chordoma cell when eEF2K is silenced. Ashour et al. showed that silencing of eEF2K in pancreatic cancer cells suppressed EMT and reduced metastasis and invasion [38]. Previously we have addressed genes that regulate the invasiveness of chordoma cells and reported that TWIST -a prominent marker of Epithelial Mesenchymal Transition (EMT)- also underlies invasiveness of chordoma [55]. In a similar study, Xie et al. delineated the molecular switches that control the metastatic capabilities of breast and lung cancer cells through suppression of eEF2K suggesting EMT-modulatory roles of eEF2K loss [36]. Therefore, we investigated the impact of eEF2K silencing on the metastasis and invasion capacities of the chordoma cells in the current study. Similar to the results in the lung [31] and breast cancer study [24], our data also point out that upon eEF2K suppression, a significant reduction in metastasis and invasion capabilities both MUG-Chor1 and UM-Chor1 cells is observed.\u003c/p\u003e \u003cp\u003eChordoma is a rare type of bone tumor and yet classiffied as often presenting with malignant properties, including recurrence and ability for metastasis. Chordoma is also highly resistant to conventional drug-based treatment methods and therefore requires new strategies that can improves the clinical outcome. Proliferation of the cells are largely depend on the proper protein synthesis, but under suboptimal conditions, proliferation needs to be blocked until the environmental conditions go back to normal. EEF2K is an enzyme that negatively regulates the protein synthesis by phosphorylating eEF2 and causes an inhibition. When cells are exposed to energy depletion eEF2K becomes activated and trigger a reduction in overall translational rates. This mechanism is also adapted by the tumor cells as they are faced with various stress conditions including nutrient deprivation, high acidity and low oxygen levels in their poorly vascularized microenviroment, which account for these harsh living conditions as a consequence of exponential growth in the tumor mass. Chordoma cells express high eEF2K levels that might explain the slow proliferating nature of the cells. By inhibiting eEF2K we were able to force the cells to become more sensitive Cisplatin and Etoposide. Upon eEF2K depletion the chordoma cells significantly lost the metastatic and invasive capacities, which holds a promise for future preclinical studies and clinical trials.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e: We would like to thank Fikrettin Sahin for kindly providing the chemicals and laboratory infrastructure to accomplish the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance with Ethical Standards\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis article does not contain any studies with human participants and/or animal participants performed by any of the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e EA, DT and ET collected and analyzed data. EA and AAHD wrote the manuscript. OFB and FS reviewed and revised the manuscript. EA supervised the entire processes. All authors read and approved the final manuscript\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest/Competing interests\u003c/strong\u003e\u0026nbsp; The authors declare that they have no competing of interest\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e All authors read and approved the final manuscript\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eChugh R, Tawbi H, Lucas DR, et al (2007) Chordoma: the nonsarcoma primary bone tumor. Oncologist 12:1344\u0026ndash;50. https://doi.org/10.1634/theoncologist.12-11-1344\u003c/li\u003e\n\u003cli\u003eBell D, Raza SM, Bell AH, et al (2016) Whole-transcriptome analysis of chordoma of the skull base. Virchows Arch 469:439\u0026ndash;449. https://doi.org/10.1007/s00428-016-1985-y\u003c/li\u003e\n\u003cli\u003eChambers KJ, Lin DT, Meier J, et al (2014) Incidence and survival patterns of cranial chordoma in the United States. Laryngoscope 124:1097\u0026ndash;1102. https://doi.org/10.1002/lary.24420\u003c/li\u003e\n\u003cli\u003eCampbell RG, Prevedello DM, Ditzel Filho L, et al (2015) Contemporary management of clival chordomas. Curr Opin Otolaryngol Head Neck Surg 23:153\u0026ndash;161. https://doi.org/10.1097/MOO.0000000000000140\u003c/li\u003e\n\u003cli\u003eLittman L, Reviews N (1856) Fletcher,14 Mabrey,28. 80\u0026ndash;90\u003c/li\u003e\n\u003cli\u003eHealey JH, Lane JM (1989) Chordoma: a critical review of diagnosis and treatment. Orthop Clin North Am 20:417\u0026ndash;426\u003c/li\u003e\n\u003cli\u003eMcMaster ML, Goldstein AM, Bromley CM, et al (2001) Chordoma: incidence and survival patterns in the United States, 1973-1995. Cancer Causes Control 12:1\u0026ndash;11. https://doi.org/10.1023/a:1008947301735\u003c/li\u003e\n\u003cli\u003eVujovic S, Henderson S, Presneau N, et al (2006) Brachyury, a crucial regulator of notochordal development, is a novel biomarker for chordomas. J Pathol 209:157\u0026ndash;65. https://doi.org/10.1002/path.1969\u003c/li\u003e\n\u003cli\u003eBailey CS, Fisher CG, Boyd MC, Dvorak MFS (2006) En bloc marginal excision of a multilevel cervical chordoma. Case report. J Neurosurg Spine 4:409\u0026ndash;414. https://doi.org/10.3171/spi.2006.4.5.409\u003c/li\u003e\n\u003cli\u003eCarrabba G, Dehdashti AR, Gentili F (2008) Surgery for clival lesions: open resection versus the expanded endoscopic endonasal approach. Neurosurg Focus 25:E7. https://doi.org/10.3171/FOC.2008.25.12.E7\u003c/li\u003e\n\u003cli\u003eGulluoglu S, Turksoy O, Kuskucu A, et al (2016) The molecular aspects of chordoma. Neurosurg Rev 39:185\u0026ndash;96; discussion 196. https://doi.org/10.1007/s10143-015-0663-x\u003c/li\u003e\n\u003cli\u003eBr\u0026uuml;derlein S, Sommer JB, Meltzer PS, et al (2010) Molecular characterization of putative chordoma cell lines. Sarcoma 2010:630129. https://doi.org/10.1155/2010/630129\u003c/li\u003e\n\u003cli\u003eHsu W, Mohyeldin A, Shah SR, et al (2011) Generation of chordoma cell line JHC7 and the identification of Brachyury as a novel molecular target. J Neurosurg 115:760\u0026ndash;9. https://doi.org/10.3171/2011.5.JNS11185\u003c/li\u003e\n\u003cli\u003eRinner B, Froehlich EV, Buerger K, et al (2012) Establishment and detailed functional and molecular genetic characterisation of a novel sacral chordoma cell line, MUG-Chor1. Int J Oncol 40:443\u0026ndash;451. https://doi.org/10.3892/ijo.2011.1235\u003c/li\u003e\n\u003cli\u003eBallard DJ, Peng H-Y, Das JK, et al (2021) Insights Into the Pathologic Roles and Regulation of Eukaryotic Elongation Factor-2 Kinase. Front Mol Biosci 8:727863. https://doi.org/10.3389/fmolb.2021.727863\u003c/li\u003e\n\u003cli\u003eBhat M, Robichaud N, Hulea L, et al (2015) Targeting the translation machinery in cancer. Nat Rev Drug Discov 14:261\u0026ndash;278. https://doi.org/10.1038/nrd4505\u003c/li\u003e\n\u003cli\u003eWang X, Xie J, Proud CG (2017) Eukaryotic Elongation Factor 2 Kinase (eEF2K) in Cancer. Cancers (Basel) 9:. https://doi.org/10.3390/cancers9120162\u003c/li\u003e\n\u003cli\u003eWang X, Regufe da Mota S, Liu R, et al (2014) Eukaryotic elongation factor 2 kinase activity is controlled by multiple inputs from oncogenic signaling. Mol Cell Biol 34:4088\u0026ndash;4103. https://doi.org/10.1128/MCB.01035-14\u003c/li\u003e\n\u003cli\u003eLeprivier G, Rotblat B, Khan D, et al (2015) Stress-mediated translational control in cancer cells. Biochim Biophys Acta 1849:845\u0026ndash;860. https://doi.org/10.1016/j.bbagrm.2014.11.002\u003c/li\u003e\n\u003cli\u003eSilvera D, Formenti SC, Schneider RJ (2010) Translational control in cancer. Nat Rev Cancer 10:254\u0026ndash;266. https://doi.org/10.1038/nrc2824\u003c/li\u003e\n\u003cli\u003eRoux PP, Topisirovic I (2012) Regulation of mRNA translation by signaling pathways. Cold Spring Harb Perspect Biol 4:. https://doi.org/10.1101/cshperspect.a012252\u003c/li\u003e\n\u003cli\u003eMendoza MC, Er EE, Blenis J (2011) The Ras-ERK and PI3K-mTOR pathways: cross-talk and compensation. Trends Biochem Sci 36:320\u0026ndash;328. https://doi.org/10.1016/j.tibs.2011.03.006\u003c/li\u003e\n\u003cli\u003eZhang Y, Cheng Y, Zhang L, et al (2011) Inhibition of eEF-2 kinase sensitizes human glioma cells to TRAIL and down-regulates Bcl-xL expression. Biochem Biophys Res Commun 414:129\u0026ndash;134. https://doi.org/10.1016/j.bbrc.2011.09.038\u003c/li\u003e\n\u003cli\u003eTekedereli I, Alpay SN, Tavares CDJ, et al (2012) Targeted silencing of elongation factor 2 kinase suppresses growth and sensitizes tumors to doxorubicin in an orthotopic model of breast cancer. PLoS One 7:e41171. https://doi.org/10.1371/journal.pone.0041171\u003c/li\u003e\n\u003cli\u003ePresneau N, Shalaby A, Idowu B, et al (2009) Potential therapeutic targets for chordoma: PI3K/AKT/TSC1/TSC2/mTOR pathway. Br J Cancer 100:1406\u0026ndash;1414. https://doi.org/10.1038/sj.bjc.6605019\u003c/li\u003e\n\u003cli\u003eMagnaghi P, Salom B, Cozzi L, et al (2018) Afatinib Is a New Therapeutic Approach in Chordoma with a Unique Ability to Target EGFR and Brachyury. Mol Cancer Ther 17:603\u0026ndash;613. https://doi.org/10.1158/1535-7163.MCT-17-0324\u003c/li\u003e\n\u003cli\u003eJiang S-X, Qi B, Yao W-J, et al (2017) Berberine displays antitumor activity in esophageal cancer cells in vitro. World J Gastroenterol 23:2511\u0026ndash;2518. https://doi.org/10.3748/wjg.v23.i14.2511\u003c/li\u003e\n\u003cli\u003eIrazoqui AP, Gonzalez A, Buitrago C (2022) Effects of calcitriol on the cell cycle of rhabdomyosarcoma cells. J Steroid Biochem Mol Biol 222:106146. https://doi.org/https://doi.org/10.1016/j.jsbmb.2022.106146\u003c/li\u003e\n\u003cli\u003eOzcan G, Ozpolat B, Coleman RL, et al (2015) Preclinical and clinical development of siRNA-based therapeutics. Adv Drug Deliv Rev 87:108\u0026ndash;119. https://doi.org/10.1016/j.addr.2015.01.007\u003c/li\u003e\n\u003cli\u003eHannon GJ, Rossi JJ (2004) Unlocking the potential of the human genome with RNA interference. Nature 431:371\u0026ndash;378. https://doi.org/10.1038/nature02870\u003c/li\u003e\n\u003cli\u003eZhang B, Zou J, Zhang Q, et al (2021) Progress in the Development of Eukaryotic Elongation Factor 2 Kinase (eEF2K) Natural Product and Synthetic Small Molecule Inhibitors for Cancer Chemotherapy. Int J Mol Sci 22:. https://doi.org/10.3390/ijms22052408\u003c/li\u003e\n\u003cli\u003eCarlberg U, Nilsson A, Nyg\u0026aring;rd O (1990) Functional properties of phosphorylated elongation factor 2. Eur J Biochem 191:639\u0026ndash;645. https://doi.org/10.1111/j.1432-1033.1990.tb19169.x\u003c/li\u003e\n\u003cli\u003eWang X, Li W, Williams M, et al (2001) Regulation of elongation factor 2 kinase by p90(RSK1) and p70 S6 kinase. EMBO J 20:4370\u0026ndash;4379. https://doi.org/10.1093/emboj/20.16.4370\u003c/li\u003e\n\u003cli\u003ePott LL, Hagemann S, Reis H, et al (2017) Eukaryotic elongation factor 2 is a prognostic marker and its kinase a potential therapeutic target in HCC. Oncotarget 8:11950\u0026ndash;11962. https://doi.org/10.18632/oncotarget.14447\u003c/li\u003e\n\u003cli\u003eJiang M, Qi L, Jin K, et al (2021) eEF2K as a novel metastatic and prognostic biomarker in gastric cancer patients. Pathol - Res Pract 225:153568. https://doi.org/https://doi.org/10.1016/j.prp.2021.153568\u003c/li\u003e\n\u003cli\u003eXie J, Shen K, Lenchine R V, et al (2018) Eukaryotic elongation factor 2 kinase upregulates the expression of proteins implicated in cell migration and cancer cell metastasis. Int J cancer 142:1865\u0026ndash;1877. https://doi.org/10.1002/ijc.31210\u003c/li\u003e\n\u003cli\u003eZhu H, Song H, Chen G, et al (2017) eEF2K promotes progression and radioresistance of esophageal squamous cell carcinoma. Radiother Oncol J Eur Soc Ther Radiol Oncol 124:439\u0026ndash;447. https://doi.org/10.1016/j.radonc.2017.04.001\u003c/li\u003e\n\u003cli\u003eAshour AA, Abdel-Aziz A-AH, Mansour AM, et al (2014) Targeting elongation factor-2 kinase (eEF-2K) induces apoptosis in human pancreatic cancer cells. Apoptosis 19:241\u0026ndash;258. https://doi.org/10.1007/s10495-013-0927-2\u003c/li\u003e\n\u003cli\u003eCheng Y, Ren X, Zhang Y, et al (2013) Integrated regulation of autophagy and apoptosis by EEF2K controls cellular fate and modulates the efficacy of curcumin and velcade against tumor cells. Autophagy 9:208\u0026ndash;219. https://doi.org/10.4161/auto.22801\u003c/li\u003e\n\u003cli\u003eXie C-M, Liu X-Y, Sham KWY, et al (2014) Silencing of EEF2K (eukaryotic elongation factor-2 kinase) reveals AMPK-ULK1-dependent autophagy in colon cancer cells. Autophagy 10:1495\u0026ndash;1508. https://doi.org/10.4161/auto.29164\u003c/li\u003e\n\u003cli\u003eFaller WJ, Jackson TJ, Knight JR, et al (2015) mTORC1-mediated translational elongation limits intestinal tumour initiation and growth. Nature 517:497\u0026ndash;500. https://doi.org/10.1038/nature13896\u003c/li\u003e\n\u003cli\u003eXiao M, Xie J, Wu Y, et al (2020) The eEF2 kinase-induced STAT3 inactivation inhibits lung cancer cell proliferation by phosphorylation of PKM2. Cell Commun Signal 18:25. https://doi.org/10.1186/s12964-020-0528-y\u003c/li\u003e\n\u003cli\u003eBircan HA, Gurbuz N, Pataer A, et al (2018) Elongation factor-2 kinase (eEF-2K) expression is associated with poor patient survival and promotes proliferation, invasion and tumor growth of lung cancer. Lung Cancer 124:31\u0026ndash;39. https://doi.org/10.1016/j.lungcan.2018.07.027\u003c/li\u003e\n\u003cli\u003eLiao Y, Chu H-P, Hu Z, et al (2016) Paradoxical Roles of Elongation Factor-2 Kinase in Stem Cell Survival*. J Biol Chem 291:19545\u0026ndash;19557. https://doi.org/https://doi.org/10.1074/jbc.M116.724856\u003c/li\u003e\n\u003cli\u003eComert Onder F, Siyah P, Durdagi S, et al (2022) Novel etodolac derivatives as eukaryotic elongation factor 2 kinase (eEF2K) inhibitors for targeted cancer therapy. RSC Med Chem 13:840\u0026ndash;849. https://doi.org/10.1039/d2md00105e\u003c/li\u003e\n\u003cli\u003eChen X, Wang K, Jiang S, et al (2022) eEF2K promotes PD-L1 stabilization through inactivating GSK3\u0026beta; in melanoma. J Immunother Cancer 10:. https://doi.org/10.1136/jitc-2021-004026\u003c/li\u003e\n\u003cli\u003eLiu L, Huang P, Wang Z, et al (2016) Inhibition of eEF-2 kinase sensitizes human nasopharyngeal carcinoma cells to lapatinib-induced apoptosis through the Src and Erk pathways. BMC Cancer 16:813. https://doi.org/10.1186/s12885-016-2853-5\u003c/li\u003e\n\u003cli\u003eGuan Y, Jiang S, Ye W, et al (2020) Combined treatment of mitoxantrone sensitizes breast cancer cells to rapalogs through blocking eEF-2K-mediated activation of Akt and autophagy. Cell Death Dis 11:948. https://doi.org/10.1038/s41419-020-03153-x\u003c/li\u003e\n\u003cli\u003eStacchiotti S, Marrari a, Tamborini E, et al (2009) Response to imatinib plus sirolimus in advanced chordoma. Ann Oncol 20:1886\u0026ndash;94. https://doi.org/10.1093/annonc/mdp210\u003c/li\u003e\n\u003cli\u003ePeng D-J, Wang J, Zhou J-Y, Wu GS (2010) Role of the Akt/mTOR survival pathway in cisplatin resistance in ovarian cancer cells. Biochem Biophys Res Commun 394:600\u0026ndash;605. https://doi.org/10.1016/j.bbrc.2010.03.029\u003c/li\u003e\n\u003cli\u003eZhang C, Lei J-L, Zhang H, et al (2017) Calyxin Y sensitizes cisplatin-sensitive and resistant hepatocellular carcinoma cells to cisplatin through apoptotic and autophagic cell death via SCF \u0026beta;TrCP-mediated eEF2K degradation. Oncotarget 8:70595\u0026ndash;70616. https://doi.org/10.18632/oncotarget.19883\u003c/li\u003e\n\u003cli\u003eAydemir E, Bayrak OF, Sahin F, et al (2012) Characterization of cancer stem-like cells in chordoma. J Neurosurg 116:810\u0026ndash;20. https://doi.org/10.3171/2011.12.JNS11430\u003c/li\u003e\n\u003cli\u003eDeng G, Zeng F, He Y, et al (2022) EEF2K silencing inhibits tumour progression through repressing SPP1 and synergises with BET inhibitors in melanoma. Clin Transl Med 12:e722. https://doi.org/10.1002/ctm2.722\u003c/li\u003e\n\u003cli\u003eZhou H, Huang S (2011) Role of mTOR signaling in tumor cell motility, invasion and metastasis. Curr Protein Pept Sci 12:30\u0026ndash;42. https://doi.org/10.2174/138920311795659407\u003c/li\u003e\n\u003cli\u003eAydemir E, Kaşikci E, Coşkun\u0026ccedil;elebi B, et al (2018) The effect of TWIST silencing in metastatic chordoma cells. Turkish J Biol 42:. https://doi.org/10.3906/biy-1801-17\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Chordoma, eEF2K, migration, siRNAs","lastPublishedDoi":"10.21203/rs.3.rs-2054247/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2054247/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground \u003c/strong\u003eEukaryotic elongation factor 2 kinase (eukaryotic elongation factor 2 kinase, eEF2K) is a calcium calmodulin dependent protein kinase that keeps the highest energy consuming cellular process of protein synthesis under check through negative regulation. EEF2K pauses global protein synthesis rates at the translational elongation step by phosphorylating its only kown substrate elongation factor 2 (eEF2), a unique translocase activity in ekaryotic cells enabling the polypeptide chain elongation. Therefore, eEF2K is thought to preserve cellular energy pools particularly upon acute development of cellular stress conditions such as nutrient deprivation, hypoxia, or infections. Recently, high expression of this enzyme has been associated with poor prognosis in an array of solid tumor types. Therefore, in a growing number of studies tremendous effort is being directed to the development of treatment methods aiming to suppress eEF2K as a novel therapeutic approach in the fight against cancer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods \u003c/strong\u003eIn our study, we aimed to investigate the changes in the tumorigenicity of chordoma cells in presence of gene silencing for eEF2K. Taking a transient gene silencing approach using siRNA particles, eEF2K gene expression was suppressed in chordoma cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003eSilencing eEF2K expression was associated with a slight increase in cellular proliferation and a decrease in death rates. Furthermore, no alteration in the sensitivity of chordoma cells to chemotherapy was detected in response to the decrease in eEF2K expression which intriguingly promoted suppression of cell migratory and invasion related properties.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur findings indicate that the loss of eEF2K expression in chordoma cell lines results in the reduction of metastatic capacity.\u003c/p\u003e","manuscriptTitle":"Impact of Silencing eEF2K Expression on the Malignant Properties of Chordoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-09-19 20:33:18","doi":"10.21203/rs.3.rs-2054247/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2022-10-07T00:50:46+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-09-16T09:43:54+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-09-13T14:18:22+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Biology Reports","date":"2022-09-13T02:44:19+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"44eb85ec-350d-4059-9670-953b1148870f","owner":[],"postedDate":"September 19th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-10-16T18:35:32+00:00","versionOfRecord":{"articleIdentity":"rs-2054247","link":"https://doi.org/10.1007/s11033-023-08257-z","journal":{"identity":"molecular-biology-reports","isVorOnly":false,"title":"Molecular Biology Reports"},"publishedOn":"2023-01-18 18:24:19","publishedOnDateReadable":"January 18th, 2023"},"versionCreatedAt":"2022-09-19 20:33:18","video":"","vorDoi":"10.1007/s11033-023-08257-z","vorDoiUrl":"https://doi.org/10.1007/s11033-023-08257-z","workflowStages":[]},"version":"v1","identity":"rs-2054247","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2054247","identity":"rs-2054247","version":["v1"]},"buildId":"-HB7Z8yhvgn0wM9Nzuekk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}