Flightless I as a molecular target to inhibit radiation-induced colorectal cancer metastasis.

Flightless I as a molecular target to inhibit radiation-induced colorectal cancer metastasis. | 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 Article Flightless I as a molecular target to inhibit radiation-induced colorectal cancer metastasis. Amirah Alhusna Mohd Yusoff, Nurul Akmaryanti Abdullah, Tengku Ahbrizal Farizal Tengku Ahmad, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4637992/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Exposure to radiation is expected to inhibit the proliferation of cancer cells, but sometimes cells are resistant to ionizing radiation. This condition can lead to cancer cell metastasis and recurrence. The mechanisms leading to the development of radioresistance are not yet fully understood. Therefore, this study aims to determine the involvement of a cytoskeletal protein, Flightless I (FliI), in cancer progression and to assess the effect of radiation exposure on the role and functionality of FliI in HCT116 cells. The expression of FliI was measured in HCT116 cells, transfected with siRNA to reduce FliI activity, and validated by Western Blot. The colony formation assay revealed a significant difference in the number of cells forming colonies on FliI-silenced cells following exposure to 6 Gy radiation. Transwell migration and invasion assays shows that silencing FliI in HCT116 cells make the m less able to migrate and invade. Further investigation via a gelatin degradation assay revealed a significant reduction in the number of cells forming invadopodia in FliI-silenced HCT116 cells compared to controls. We proved the efficacy of FliI in inhibiting radiation-enhanced cancer migration and invasion, indicating its potential as a therapeutic target to enhance radiosensitization in CRC patients. Biological sciences/Cancer Biological sciences/Cell biology Biological sciences/Molecular biology Health sciences/Medical research Health sciences/Oncology Flightless I ionizing radiation migration and invasion colorectal cancer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION Colorectal cancer (CRC) is one of the most common cancers and the third leading cause of cancer related death in the world ( 1 ). Its incidence has consistently grown in recent years, especially in developing countries, due to shifts in lifestyle patterns, physical activity and diet ( 2 ). It has been reported that the five-year survival rate is increased when CRC is detected at an early stage. However, the survival rate is reduced when the cancer progresses to an advanced stage, including metastasis and recurrence ( 2 ). One of the main cancer treatments is radiotherapy, which targets and damages cancer cells using high-energy of ionizing radiation ( 3 ). As a result, DNA structure will be damaged, affecting the survival of the tumours and subsequently interfering with the capacity of cancer cells to proliferate ( 4 ). Previous studies have shown that radiotherapy may facilitate the invasion and metastatic processes. Although radiation therapy has consistently proven to be effective in treating various cancer types, there are some cases of metastases that are possible due to the resistance of cancer cells to radiation ( 5 , 6 , 7 ). Migration and invasion are fundamental processes in cancer development, playing a critical role in the metastatic spread of malignant cells. Metastasis can be characterized by the spread of cancer cells from the primary tumor to other tissues or organs in the body through the blood or lymphatic system. This complex process is known as the metastatic cascade, which involves the sequential steps of tumour cell dissociation from the primary tumour, penetration of tissue barriers, invasion into adjacent tissues, intravasation into the bloodstream and lymphatic vessels, and subsequently extravasation into secondary sites ( 8 ). However, previous studies have provided circumstantial evidence associating ionizing radiation with the promotion of migration and invasion in cancer cells, particularly through mechanisms such as epithelial mesenchymal transition (EMT) ( 9 , 10 ), interaction with the stromal environment ( 11 , 12 ), and modulation of matrix-degrading metalloproteinases (MMPs) ( 13 , 14 , 15 ). This process involves the actin cytoskeleton, which is essential for maintaining cell structure, facilitating movement, and regulating intracellular transport. During malignant transformation, the cytoskeleton network can be reprogrammed to aid in cancer progression 16). Flightless I (FliI) is an actin-binding member of the gelsolin family known for its role in inhibiting actin polymerization. According to Cowin et al. (2012) and He et al. (2018), this protein has been linked to a variety of biological and physiological processes, such as cell migration, proliferation, and the progression of cancer ( 17 , 18 ). FliI has been widely studied in cancer cells progression, and some of them suggest that upregulation of FliI can promote proliferation, inhibit apoptosis, and enhance cell invasion ( 19 , 20 , 21 ). On the other hand, a lower level of FliI is linked to less invasion, which slow down the growth and activation of cancer cells ( 19 , 21 ). Although previous research has explored the role of FliI in cancer cells, its role in irradiated CRC remains poorly understood. The aim of the study is to determine whether FliI has a specific role in the activation and progression of irradiated FliI in the CRC cell line. We anticipate these findings to unveil the regulatory mechanism of FliI in irradiated CRC. MATERIALS AND METHODS Cell lines and culture conditions The human colorectal carcinoma cell line (HCT116) was purchased from the American Type Culture Collection (ATCC, USA) and routinely cultured in McCoy’s 5A medium (Cytiva, USA) containing 10% fetal bovine serum (FBS; Hyclone, USA) and 5% Penicillin-streptomycin solution in a humidified atmosphere of 5% CO2 at 37°C. When cell reached 80–90% confluence, cells were detached with 5 mM trypsin/Ethylenediaminetetraacetic acid (EDTA), then re-suspended in culture media. Viable cells were counted using a hemocytometer with trypan blue staining. Transient siRNA transfection HCT116 cells were transfected with small interfering RNA (siRNA) targeting FliI (siFliI), using Lipofectamine™ RNAiMAX transfection reagent (Invitrogen; ThermoFisher Scientific, Inc.), following the manufacturer’s recommendations. The cells were transfected for 48 hours for further analysis. Western Blot was done to validate this transfection. A knockdown efficiency of 70% or greater is considered effective and acceptable. X-ray irradiation X-ray generator (ISOVOLT 225 M2) in Division of Industrial Technology, Malaysian Nuclear Agency was set at 200kVp and 13 mA. Irradiation is made through a copper and aluminium filter with a thickness of 0.5 mm and a dose rate of 1Gy/min. Clonogenic Assay The cells were transfected with siRNA before X-ray irradiation with various doses (0, 2, 4, and 6 Gy) and seeded in 6-well plates. Following irradiation, cells were immediately trypsinized and plated for clonogenic survival. After 10 days, the colonies were fixed with ice cold absolute ethanol and stained with 0.1% crystal violet. Colonies of at least 50 cells were scored as survivors and counted with naked eyes or stereoscopic microscope. Plating efficiency (PE) was calculated with the following formula: $$\text{P}\text{E}= \frac{Number of colonies counted}{Number of cells seeded} \times 100$$ The survival fraction (SF) was calculated with the following formula: $$\text{S}\text{F}= \frac{Number of colonies counted}{Number of cells seeded \times \left(\text{P}\text{E}/100\right)}$$ To evaluate the degree of radiosensitization, the sensitizer enhancement ratio (SER) was determined by dividing the survival fraction at a 2 Gy of irradiation dose for cells without FliI-targeted transfection by the survival fraction at the 2 Gy of irradiation dose for cells with FliI-targeted transfection. Dose-response curves were graphed on a log-linear plot to illustrate the relationship with radiation dose. To model survival curves using the linear-quadratic (LQ) with an equation of \(S=\text{exp}\left[-\left(\alpha D+{\beta D}^{2}\right)\right]\) , the Clonogenic Survival Calculation Software (CS-CAL), developed by the Translational Radiation Oncology Group at the German Cancer Research Centre, was employed. The software is accessible online at http://angiogenesis.dkfz.de/oncoexpress/software/cs-cal/ ( 22 ). Transwell Migration and Invasion Assay Transwell migration and invasion assay were carried out using cell culture inserts with pore size of 8µm in 24-well plates (Corning, USA). Matrigel (Corning, USA) was diluted with serum-free medium from 8.9 mg/mL (stock solution) to 0.1 mg/mL (working concentration) and coated at the top chamber of the cell culture inserts for invasion assay. On the other hand, matrigel coating was not applied to the inserts for the transmigration assay. Approximately 5.0 × 10 4 cells/mL of irradiated and non-irradiated transfected cells were plated in the upper chamber filled with medium containing 1% of serum. The lower chamber was filled with medium containing 20% of serum as chemoattractant. After 24h of incubation at 37°C, the cells were fixed in ice cold absolute ethanol and stained with crystal violet dye (Sigma-Aldrich, USA). Images were captured at twelve random fields at 200× magnification using inverted microscope. Number of cells migrated or invaded through a porous membrane, either coated or non-coated with Matrigel were calculated. Gelatin Degradation Assay Coverslips were coated with 50 µg/ml of 488 Oregon Green Gelatin (Sigma-Aldrich) and irradiated and non-irradiated transfected cells were seeded on top of the coated coverslip. After 48 hours incubation, cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Cells were then stained with rhodamine-phalloidin (1:100) and Hoechst (1:1000). The coverslips were mounted on glass slides and let dried overnight at 4 o C. To quantify invadopodia, 100 cells were randomly chosen from each coverslip and observed under a fluorescence microscope. The average percentage of cells displaying invadopodia was then calculated ( 23 , 24 ). $$\% of cells with invadopodia=\frac{Total number of cells with invadopodia}{Total number of cells} \times 100$$ Western blot Immunoblotting was conducted using whole cell lysates. Irradiated and non-irradiated transfected cells were rinsed with cold PBS, lysed with RIPA buffer supplemented with protease inhibitor (Thermo Scientific, USA). Protein concentrations were determined using the Pierce™ BCA Protein Assay Kit (Thermo Scientific, USA). Equal amounts of protein were separated by SDS-PAGE and transferred to a nitrocellulose membrane (Azure Biosystems, USA). The membranes were blocked in TBST with 5% non-fat skim milk at room temperature for 1 hour and incubated with primary antibodies overnight at 4°C. Subsequently, the membranes were probed with an appropriate horseradish peroxidase-linked (HRP) secondary antibody. Chemiluminescence was generated using Western Bright ECL HRP substrate (Advansta, USA). Antibodies against Flightless I (1:1000), GAPDH (1:5000), and HRP-linked goat anti-rabbit secondary antibodies (1:3000) were obtained from Cell Signalling Technology (MA, USA), antibodies against MMP2 (1:1000) was bought from Elabscience (Texas, USA) and antibodies against Cortactin (1:1000) was bought from Abcam (Cambridge, UK). ImageJ analysis software was used for densitometry, where intensity of the samples was standardized to the intensity of GAPDH. Statistical Analysis All data presented are representative of at least three independent experiments. Results are expressed as means ± SEM, where groups were compared by ANOVA. Statistical analysis was performed using Graph Pad Prism Version 9.5.1 software and p < 0.05 was considered as statistically significant. RESULTS Silencing the FliI Gene Increases Radiosensitivity in Irradiated HCT116 Cells Clonogenic survival assays were conducted to determine whether knockdown of FliI could enhance radiosensitization in HCT116. Wild-type (WT), non-targeting siRNA (siControl) and siFliI transfected cells were exposed to various doses of ionizing radiation (0 to 6 Gy). In the absence of transfection, significant reduction in number of colonies formed were observed in all cells with increasing doses of X-ray (Fig. 1; p < 0.001). Interestingly, following reduced expression of FliI (siFliI), HCT116 cells become more sensitive to ionizing radiation particularly at 6 Gy (Fig. 1B; p < 0.05) compared to WT and siControl of HCT116 cells. The survival fraction at 6 Gy (SF6) of siControl cells was reduced from 0.00025 to 0.00012 in the siFliI cells (Fig. 1B). The sensitiser enhancement ratio (SER) of silenced FliI compared to WT and siControl at 2, 4 and 6 Gy were ranging from 1.15 to 2.06 (Table 1 ). A 0 Gy 2 Gy 4 Gy 6 Gy B WT siControl siFliI Seeding density 100 1000 2000 10000 Figure 1: Effects of FliI silencing on radiosensitivity HCT116 cells by clonogenic assay. (A) Cell colony formation of HCT116 cell after exposed to X-ray at different doses. (B) The survival curves of WT, siControl and siFliI for HCT116 cells were plotted using the LQ model, after exposed to 0 Gy, 2 Gy, 4 Gy and 6 Gy of X-ray. Results represent mean ± SEM of three experiments with *p < 0.05 and ***p < 0.001. Table 1 Sensitizer Enhancement Ratio (SER) of transfected HCT116 cells (siFliI) against WT and siControl at 2, 4 and 6 Gy dose of irradiation. SER WT/siFliI siControl/siFliI SF2 1.155 1.225 SF4 1.49 1.58 SF6 1.78 2.06 Silencing the FliI Gene Inhibits Migration and Invasion of HCT116 Cells The migration potential of HCT116 cells with a silenced FliI gene against irradiation was investigated using the transwell migration technique (Fig. 2A). Reduced expression of FliI gene did not significantly reduce the migratory ability of the cells (Fig. 2B; p > 0.05). However, exposure to ionizing radiation at 2, 4, and 6 Gy significantly inhibit the migratory ability of HCT116 cells. The number of migrated cells was significantly lower when FliI was knocked down and radiation was applied compared to WT and siControl (Fig. 2B; p < 0.05). Additionally, the invasive potential of FliI gene silencing cells in response to irradiation was evaluated using a transwell invasion experiment, where the extracellular matrix environment was stimulated by coating the transwell with Matrigel (Fig. 2C). Reduced FliI expression markedly impeded the invasive potential of non-irradiated cells (Fig. 2D; p < 0.05). Upon exposure to radiation, there was a significant reduction in the number invading HCT116 cells that were transfected with siFliI compared to non-transfected and siControl (Fig. 2D; p < 0.05). A 0 Gy 2 Gy 4 Gy 6 Gy B WT siControl siFliI C 0 Gy 2 Gy 4 Gy 6 Gy D WT siControl siFliI Figure 2: FliI silencing significantly decreased migration and invasion of irradiated HCT116 cells. (A) Representative images of transwell migration membranes stained with 0.1% crystal violet at a magnification of 200x. (B) Graphical representative of total number of migrated cells for FliI-silenced cells compared to WT and siControl of HCT116 cells. Results represent mean ± SEM of three experiments with *p < 0.05. Scale bars = 200µm (C) Representative images of transwell invasion membranes stained with 0.1% crystal violet at a magnification of 200x. (B) Graphical representative of total number of invaded cells for FliI-silenced cells compared to WT and siControl of HCT116 cells. Results represent mean ± SEM of three experiments with *p < 0.05 and **p < 0.01. Silencing the FliI Gene Decreases Radiation-Induced Invadopodia-Formation in HCT116 cells. The role of FliI expression in HCT116 cells in inhibiting invadopodia formation was investigated. Invadopodia are dynamic membrane extensions that facilitate the degradation of the extracellular matrix, contributing to cell invasion. They were detected through regions of matrix degradation, distinguished by the absence of green fluorescence in the gelatin and exhibiting co-localization with red actin spots. The blue staining (Hoechst) clearly showed the nuclei of the cells (Fig. 3A). The effect of FliI knockdown was investigated on the percentage of cells forming invadopodia in both non-irradiated and irradiated HCT116 cells. Knockdown of the FliI gene was shown to significantly inhibit invadopodia formation (Fig. 3B; p < 0.05). Following exposure to a 2 Gy of X-ray radiation, significant increase in the percentage of cells forming invadopodia were observed compared to non-irradiated WT, siControl, and siFliI (Fig. 3B; p < 0.05). Similar to non-irradiated HCT116 cells, irradiated HCT116 cells with decreased FliI expression exhibit a notable reduction in invadopodia formation, in contrast to WT and siControl (Fig. 3B; p < 0.001). Merged FITC Gelatin Rhodamine Phalloidin Hoechst WT siControl siFliI WT + 2Gy siControl + 2Gy siFliI + 2Gy B FliI Expression in HCT116 Cells is Upregulated with Ionizing Radiation The effect of ionizing radiation on FliI expression was determined by Western Blot analysis (Fig. 4A). Interestingly, FliI expression increased significantly with increasing of X-ray dose (Fig. 4B; p < 0.05). The highest FliI expression level was observed at 6 Gy of X-ray (p < 0.01). A 0 Gy 2 Gy 4 Gy 6 Gy B FliI 145 kDa GAPDH 36 kDa Figure 4: FliI expression is upregulated in irradiated HCT116 cells. (A) Western blot analysis of FliI protein in HCT116 cells upon exposed to 0 Gy, 2 Gy, 4 Gy and 6 Gy of X-ray. (B) Graphical representation of relative expression of FliI in HCT116 cells from Western Blot analysis. Results represent mean ± SEM of three independent experiments with *p < 0.05 and **p < 0.01. The Effects of Decreased FliI Expression and Post-Irradiation on the Protein Expression levels of Beta Catenin, Cortactin and MMP2. The effectiveness of FliI siRNA transfection was assessed by Western Blot analysis, which revealed a significant reduction in FliI expression in HCT116 cells, resulting in estimated 80–90% reduction in FliI activity (Fig. 5 B; p < 0.05). Reduced FliI expression has been shown to hinder invasion by transwell assays and gelatin degradation. Nevertheless, the specific molecular player linked to this activity has not been determined. Therefore, we selected three specific proteins, namely β-Catenin, cortactin, and MMP2, to examine their involvement in the FliI signalling pathway (Fig. 5 A). After exposure to a radiation dose of 2 Gy, the level of MMP2 expression showed a little increase, although this change did not have statistical significance (Fig. 5 E; p > 0.05). In addition, a notable drop in FliI led to a modest reduction in β-Catenin expression in both non-irradiated and irradiated HCT116 cells (Fig. 5 C). However, this decrease did not reach statistical significance (p > 0.05). The decrease in FliI expression did not have a significant impact on the expression levels of cortactin and MMP2 (Fig. 5 D and Fig. 5 E; p > 0.05). A FliI 145 kDa β-cat 94 kDa Cortactin 80 kDa MMP2 72 kDa GAPDH 36 kDa B C D E DISCUSSION The three cell lines used in this study (MU41, U87MG and LN229) form functional invadopodia that degrade the FITC-labelled gelatin which can be seen to co-localize with rhodamine phalloidin-stained actin puncta (Fig.1a). The Radiotherapy is a very effective treatment against many types of cancer, which involves the use of high-energy radiation to target and eradicate cancer cells ( 25 ). This process disrupts the ability to divide and proliferate by inducing breaks in the DNA strands. While some cells may naturally be resistant to radiation, this could be because of DNA repair mechanisms, protective microenvironments, or genetic changes ( 4 , 25 , 26 , 27 , 28 ). Despite its effectiveness, radiotherapy may have unanticipated negative effects because it paradoxically promotes the progression, migration, and invasion of cancer cells ( 5 , 29 ). Exposure to ionizing radiation can trigger survival mechanisms that result in enhanced aggressiveness of cancer cells ( 5 , 29 ). Numerous studies have been investigating the intricate relationship between the ionizing radiation and cytoskeletal proteins within various types of cancers, including CRC ( 30 , 31 ). FliI is a cytoskeletal protein that influences tumorigenesis and cancer progression. In breast cancer cells, elevated FliI levels have been associated with increased tumor invasiveness and metastasis ( 18 , 32 ). Choi et al. (2020) found that FliI acts as an inhibitor of ER-stress induced cell apoptosis in CRC cells ( 33 ). By modulating cellular Ca2 + balance, FliI reduces the apoptotic responses triggered by ER stress. Furthermore, previous studies investigated the role of FliI in skin cancer, which regulate the cell motility and tissue invasion ( 19 , 21 ). The present study aims to investigate the relationship between FliI expression and the resistance of HCT116 CRC cells to ionizing radiation. A significant increase in FliI expression was observed in irradiated HCT116 colorectal cancer cells. Then, the expression of FliI was significantly reduced through the transfection of siRNA targeting FliI. Suppression of FliI expression was shown to enhance the radiosensitivity of HCT116 cancer cells, with the highest SER at SF6 (2.06), with the value (WT and siControl) being close to the expected value from a previous study by Chen et al. (2010) ( 34 ). This result indicates that FliI plays a role in enhancing the radio-sensitization of HCT116 cells. Previously, FliIs have been demonstrated to be involved in cell motility and tissue invasion ( 19 , 21 ). The transwell migration and invasion assay indicates that ionizing radiation alone reduced the number of migrated and invaded cells, most probably due to the reduced cell viability caused by radiation itself. However, cancer cells that could resist the effects of ionizing radiation can become more invasive. As evident from the gelatin degradation assay analysis, the data obtained in this study indicate that irradiated HCT116 cells significantly increased invadopodia formation. These results are consistent with previous studies demonstrating a significant increase in invadopodia formation when glioma and glioblastoma cells were irradiated ( 35 , 36 , 37 ). Inhibition of FliI protein significantly inhibits the migration and invasion abilities of irradiated HCT116 cells. We found that reduced expression of FliI suppressed invasion by reducing the formation of invadopodia and reducing the ability of HCT116 cells to degrade gelatin. Studies have shown that ionizing irradiation can enhance the invasiveness and metastatic potential of cancer cells through various mechanisms. For instance, irradiation-induced DNA damage and cellular stress responses can trigger signaling pathways involved in epithelial-mesenchymal transition (EMT), a biological process wherein epithelial cells undergo changes leading to a mesenchymal phenotype. This transition is characterized by the loss of cell adhesion and polarity, which allows cells to acquire enhanced migratory and invasive properties ( 38 , 39 ). As cells lose their epithelial characteristics, epithelial markers like E-cadherin and cytokeratins are typically downregulated, while mesenchymal markers like N-cadherin, vimentin, and fibronectin are upregulated, reflecting the acquisition of a mesenchymal phenotype ( 40 , 41 ). Numerous studies have provided evidence supporting the idea that ionizing radiation can induce changes promoting EMT in diverse cancer types. This includes instances observed in glioblastoma, lung cancer and CRC that showed a low level of epithelial markers and a high level of mesenchymal markers upon radiation exposure ( 42 , 43 , 44 , 45 ). Additionally, ionizing irradiation promotes the expression of matrix metalloproteinases (MMPs), which enhance the invasive capability of cancer cells. Studies have shown increased MMP expression in irradiated CRC and breast cancer cells ( 13 , 15 ). In this study, irradiated HCT116 cells were found to increase the invasiveness of the cells, as the percentage of cells forming invadopodia showed a significant increase. This is in line with the upregulation of FliI protein expression in irradiated HCT116 cells as a result of ionizing radiation, which enhances the invasive characteristics of HCT116 cells. When invadopodia formation was reduced following FliI silencing, this situation led to an investigation of β-catenin, cortactin and MMP2 expression levels. β-Catenin signaling has induced metastasis in cancers such as breast cancer, hepatocellular carcinoma (HCC), and melanoma by facilitating metastatic spread through increased of cell migration and invasion ( 46 , 47 , 48 ). Furthermore, cortactin and MMP2 plays a crucial role in matrix degradation during cancer cell invasion, exhibit increased localization at invadopodia. ( 49 , 50 ). The results indicated that a significant reduction in FliI protein levels has not significantly impacted β-catenin expression, although there was a slight reduction. Previous studies have shown that when FliI is silenced, β-catenin expression reduce significantly ( 21 ). This is because of the way FliI and β-catenin are controlled. The observed discrepancy could be caused by a variety of factors, such as differences in experimental conditions, the specific cell lines used, or the degree of FliI silencing achieved. These findings also shows that cortactin and MMP2 expression remains unchanged upon FliI silencing. This suggests that the regulation of cortactin and MMP2 may be FliI-independent, thus, cortactin and MMP2 are transported and targeted via different molecular mechanisms. In addition, this independent regulation might affect other types of MMPs, such as MMP9 and MMP14, which are also involved in invadopodia formation, particularly in HCT116 cells ( 49 , 51 , 52 ). Further research is necessary to elucidate another actin cytoskeleton related to EMT, such as E-cadherin and vimentin, to determine the specific conditions under which FliI influences these pathways. Furthermore, studies have shown that inhibiting Flii suppresses CRC proliferation, migration, and invasion in vitro . These findings suggest that FliI may be important in the development of new treatments that will make radiotherapy more effective and lessen the impact of aggressive metastatic CRC. CONCLUSION The analysis from this study revealed a significant increase in the expression of FliI in HCT116 cells after exposure to ionizing radiation. The suppression of FliI expression resulted in increased sensitivity to radiation in HCT116 cells, along with a notable reduction in their ability to migrate and invade. Further research effort could focus on exploring alternative signalling pathways influenced by FliI, including key proteins involved in cell migration and invasion upon exposure to ionizing radiation. Additionally, assessing the influence of FliI on the epithelial-mesenchymal transition (EMT) marker post-radiation exposure could elucidate its regulatory mechanism. This investigation should use multiple colorectal cancer cell lines to ensure the results are broadly applicable and account for genetic and phenotypic variability among different cell lines, or in vivo studies using animal models to validate findings in a physiological context. This result indicate that FliI may serve as a promising target for molecular therapeutics designed to regulate the aggressiveness of cancer cells thus reducing the metastasis of CRC. Declarations Author Contribution A.A.M.Y performed the experiments, analysed the data and wrote the paper. N.M.Z. and N.F.M.H conceived and designed the experiments. N.M.Z., N.F.M.H, N.A. A. and T.A.F.T.A provided intellectual inputs and analysed the data. N.E.Q.A provides optimised methodologies and guidance for various experiments. All authors reviewed the manuscript. ACKNOWLEDGEMENT This study was financially supported by the Fundamental Research Grant Scheme (FRGS/1/2020/SKK06/UPM/02/9), Ministry of Education, Malaysia. 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Flightless I (Drosophila) homolog facilitates chromatin accessibility of the estrogen receptor α target genes in MCF-7 breast cancer cells. Biochemical and biophysical research communications 446(2), 608–613 (2014). Choi, S. S.et al. Flightless-1 inhibits ER stress-induced apoptosis in colorectal cancer cells by regulating Ca2 + homeostasis. Experimental & molecular medicine 52(6), 940–950 (2020). Chen, W. S. et al. Depletion of securin induces senescence after irradiation and enhances radiosensitivity in human cancer cells regardless of functional p53 expression. International journal of radiation oncology, biology, physics 77(2), 566–574 (2010). Whitehead, C. A. et al. Inhibition of Radiation and Temozolomide-Induced Invadopodia Activity in Glioma Cells Using FDA-Approved Drugs. Translational oncology 11(6), 1406–1418 (2018) Dinevska, M. et al. Inhibition of Radiation and Temozolomide-Induced Glioblastoma Invadopodia Activity Using Ion Channel Drugs. Cancers 12(10), 2888 (2020). Jones, D. et al. Repurposing FDA-approved drugs as inhibitors of therapy-induced invadopodia activity in glioblastoma cells. Molecular and cellular biochemistry 478(6), 1251–1267 (2023). Yang, J. Guidelines and definitions for research on epithelial-mesenchymal transition. Nature reviews: Molecular cell biology 21(6), 341–352 (2020). Ribatti, D., Tamma, R., & Annese, T. Epithelial-mesenchymal transition in cancer: a historical overview. Translational oncology 13(6), 100773 (2020). Liao, T. T., & Yang, M. H. Revisiting epithelial-mesenchymal transition in cancer metastasis: the connection between epithelial plasticity and stemness. Molecular oncology 11(7), 792–804 (2017). Zhang, N., Ng, A. S., Cai, S., Li, Q., Yang, L., & Kerr, D. Novel therapeutic strategies: Targeting epithelial–mesenchymal transition in colorectal cancer. The Lancet Oncology 22(8), e358-e368 (2021). Kawamoto, A. et al. Radiation induces epithelial-mesenchymal transition in colorectal cancer cells. Oncology reports 27(1), 51–57 (2012). Mahabir, R. et al. Sustained elevation of Snail promotes glial-mesenchymal transition after irradiation in malignant gliom. Neuro-oncology 16(5), 671–685 (2014). Jefri, M., Huang, Y. N., Huang, W. C., Tai, C. S., & Chen, W. L. YKL-40 regulated epithelial-mesenchymal transition and migration/invasion enhancement in non-small cell lung cancer. BMC cancer 15, 1–10 (2015). Zhang, J., Ding, L., Sun, G., Ning, H., & Huang, R. Suppression of LINC00460 mediated the sensitization of HCT116 cells to ionizing radiation by inhibiting epithelial-mesenchymal transition. Toxicology research 9(2), 107–116 (2020). Yook, J. I. et al. A Wnt-Axin2-GSK3beta cascade regulates Snail1 activity in breast cancer cells. Nature cell biology 8(12), 1398–1406 (2006). Liu, L. et al. Activation of beta-catenin by hypoxia in hepatocellular carcinoma contributes to enhanced metastatic potential and poor prognosis. Clinical cancer research: an official journal of the American Association for Cancer Research 16(10), 2740–2750 (2010). Damsky, W. E. et al. β-catenin signaling controls metastasis in Braf-activated Pten-deficient melanomas. Cancer cell 20(6), 741–754 (2011). Jacob, A., & Prekeris, R. The regulation of MMP targeting to invadopodia during cancer metastasis. Frontiers in cell and developmental biology 3, 4 (2015). Jeannot, P., & Besson, A. Cortactin function in invadopodia. Small GTPases 11(4), 256–270 (2020). Weng, M. T. et al. Hes1 Increases the Invasion Ability of Colorectal Cancer Cells via the STAT3-MMP14 Pathway. PloS one 10(12), e0144322 (2015). Wang, W. et al. TIMP-2 inhibits metastasis and predicts prognosis of colorectal cancer via regulating MMP-9. Cell adhesion & migration 13(1), 273–284 (2019). Additional Declarations No competing interests reported. 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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-4637992","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":330730598,"identity":"f44253cf-3f93-40ae-9160-9c437ce6bda0","order_by":0,"name":"Amirah Alhusna Mohd Yusoff","email":"","orcid":"","institution":"Universiti Putra Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Amirah","middleName":"Alhusna Mohd","lastName":"Yusoff","suffix":""},{"id":330730599,"identity":"458e2ee0-f144-4636-8b21-e6c4b55c0d8f","order_by":1,"name":"Nurul Akmaryanti Abdullah","email":"","orcid":"","institution":"Universiti Putra Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Nurul","middleName":"Akmaryanti","lastName":"Abdullah","suffix":""},{"id":330730600,"identity":"4eaf71c4-fd55-4160-bfb3-9f8ac6f8308f","order_by":2,"name":"Tengku Ahbrizal Farizal Tengku Ahmad","email":"","orcid":"","institution":"Malaysian Nuclear Agency","correspondingAuthor":false,"prefix":"","firstName":"Tengku","middleName":"Ahbrizal Farizal Tengku","lastName":"Ahmad","suffix":""},{"id":330730601,"identity":"0f6cb4a1-9790-4584-b5f7-419eb97efcbd","order_by":3,"name":"Nor Ezleen Qistina Ahmad","email":"","orcid":"","institution":"Universiti Putra Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Nor","middleName":"Ezleen Qistina","lastName":"Ahmad","suffix":""},{"id":330730602,"identity":"a417cefd-d587-4b32-864f-ad5d2979aa7d","order_by":4,"name":"Nur Fariesha Md Hashim","email":"","orcid":"","institution":"Universiti Putra Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Nur","middleName":"Fariesha Md","lastName":"Has","suffix":"Md"},{"id":330730605,"identity":"b0db02c3-3571-42e3-9197-cee9717121da","order_by":5,"name":"Noraina Muhamad Zakuan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxElEQVRIiWNgGAWjYLCCBAYGOQiLjQQtxiRqAYLEBqK1mLP3PvzwcIdd+obb3QkMH8oOM+jOSMCvxbLnuLFE4pnk3A13zm5gnHHuMIPZDQJaDG6kMUgktjHnbriRu4GZt404Lcw/Etvq0w1AWv4SqYUNaMvhBLAWRqK0nDnGZpHYdtxwJtAvB3vOpfOYnXlAQMvxNuabP9uq5flu92588KPMWs7sOAFbEECCgeEAkOJhECBFCwTwHyBWyygYBaNgFIwQAABBxEoCnjFWcwAAAABJRU5ErkJggg==","orcid":"","institution":"Universiti Putra Malaysia","correspondingAuthor":true,"prefix":"","firstName":"Noraina","middleName":"Muhamad","lastName":"Zakuan","suffix":""}],"badges":[],"createdAt":"2024-06-25 17:00:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4637992/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4637992/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":61183655,"identity":"c75eba1a-7b49-4b1d-b87b-4e64a5386ccb","added_by":"auto","created_at":"2024-07-26 17:06:01","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":61614,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of FliI silencing on radiosensitivity HCT116 cells by clonogenic assay.\u003c/strong\u003e (A) Cell colony formation of HCT116 cell after exposed to X-ray at different doses. (B) The survival curves of WT, siControl and siFliI for HCT116 cells were plotted using the LQ model, after exposed to 0 Gy, 2 Gy, 4 Gy and 6 Gy of X-ray. Results represent mean ± SEM of three experiments with *p\u0026lt;0.05 and ***p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"FIGURE1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4637992/v1/d653f5258e33b55d5e06a21a.jpg"},{"id":61182675,"identity":"1dd2a095-ef9c-42a3-a60e-e96e04c857f1","added_by":"auto","created_at":"2024-07-26 16:58:01","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":169208,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFliI silencing significantly decreased migration and invasion of irradiated HCT116 cells.\u003c/strong\u003e (A) Representative images of transwell migration membranes stained with 0.1% crystal violet at a magnification of 200x. (B) Graphical representative of total number of migrated cells for FliI-silenced cells compared to WT and siControl of HCT116 cells. Results represent mean ± SEM of three experiments with *p\u0026lt;0.05. Scale bars = 200mm (C) Representative images of transwell invasion membranes stained with 0.1% crystal violet at a magnification of 200x. (B) Graphical representative of total number of invaded cells for FliI-silenced cells compared to WT and siControl of HCT116 cells. Results represent mean ± SEM of three experiments with *p\u0026lt;0.05 and **p\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"FIGURE2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4637992/v1/a006fed0b055270a2695083b.jpg"},{"id":61183656,"identity":"7b28660d-5190-44b6-ac19-a1e02ada3658","added_by":"auto","created_at":"2024-07-26 17:06:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1919039,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"FIGURE3.png","url":"https://assets-eu.researchsquare.com/files/rs-4637992/v1/124a38240d75e611d6243c6c.png"},{"id":61182673,"identity":"611b69b7-2de8-451e-9703-f0f12e749fea","added_by":"auto","created_at":"2024-07-26 16:58:01","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":39129,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFliI expression is upregulated in irradiated HCT116 cells.\u003c/strong\u003e (A) Western blot analysis of FliI protein in HCT116 cells upon exposed to 0 Gy, 2 Gy, 4 Gy and 6 Gy of X-ray. (B) Graphical representation of relative expression of FliI in HCT116 cells from Western Blot analysis. Results represent mean ± SEM of three independent experiments with *p\u0026lt;0.05 and **p\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"FIGURE4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4637992/v1/67f3ad357981753478b4f159.jpg"},{"id":61185271,"identity":"936a2d52-75aa-4f65-ad4a-232632ee340b","added_by":"auto","created_at":"2024-07-26 17:22:01","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":448501,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of β-Catenin and MMP2 in HCT116 cells.\u003c/strong\u003e (A) Representative images of western blot analysis of FliI, β-Catenin, cortactin and MMP2 protein in HCT116 cells. (B) Graphical representation of relative expression of FliI. (C) Graphical representation of relative expression of β-Catenin. (D) Graphical representation of relative expression of Cortactin. (E) Graphical representation of relative expression of MMP2. Results represent mean ± SEM of three experiments with ***p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"FIGURE5.png","url":"https://assets-eu.researchsquare.com/files/rs-4637992/v1/df9d64179fea76a86902ab94.png"},{"id":67085631,"identity":"3e58dae8-5e97-4340-ae36-15c0106e2e6b","added_by":"auto","created_at":"2024-10-21 05:55:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3581968,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4637992/v1/1f2a3de4-e236-479f-8895-737f51c61ab3.pdf"},{"id":61184584,"identity":"daebb33c-9690-4009-8d91-f94b98276266","added_by":"auto","created_at":"2024-07-26 17:14:01","extension":"pdf","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":107881,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4637992/v1/176364b096d126fe3c42f31f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Flightless I as a molecular target to inhibit radiation-induced colorectal cancer metastasis.","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eColorectal cancer (CRC) is one of the most common cancers and the third leading cause of cancer related death in the world (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Its incidence has consistently grown in recent years, especially in developing countries, due to shifts in lifestyle patterns, physical activity and diet (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). It has been reported that the five-year survival rate is increased when CRC is detected at an early stage. However, the survival rate is reduced when the cancer progresses to an advanced stage, including metastasis and recurrence (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). One of the main cancer treatments is radiotherapy, which targets and damages cancer cells using high-energy of ionizing radiation (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). As a result, DNA structure will be damaged, affecting the survival of the tumours and subsequently interfering with the capacity of cancer cells to proliferate (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePrevious studies have shown that radiotherapy may facilitate the invasion and metastatic processes. Although radiation therapy has consistently proven to be effective in treating various cancer types, there are some cases of metastases that are possible due to the resistance of cancer cells to radiation (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Migration and invasion are fundamental processes in cancer development, playing a critical role in the metastatic spread of malignant cells. Metastasis can be characterized by the spread of cancer cells from the primary tumor to other tissues or organs in the body through the blood or lymphatic system. This complex process is known as the metastatic cascade, which involves the sequential steps of tumour cell dissociation from the primary tumour, penetration of tissue barriers, invasion into adjacent tissues, intravasation into the bloodstream and lymphatic vessels, and subsequently extravasation into secondary sites (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). However, previous studies have provided circumstantial evidence associating ionizing radiation with the promotion of migration and invasion in cancer cells, particularly through mechanisms such as epithelial mesenchymal transition (EMT) (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e), interaction with the stromal environment (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e), and modulation of matrix-degrading metalloproteinases (MMPs) (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). This process involves the actin cytoskeleton, which is essential for maintaining cell structure, facilitating movement, and regulating intracellular transport. During malignant transformation, the cytoskeleton network can be reprogrammed to aid in cancer progression 16).\u003c/p\u003e \u003cp\u003eFlightless I (FliI) is an actin-binding member of the gelsolin family known for its role in inhibiting actin polymerization. According to Cowin et al. (2012) and He et al. (2018), this protein has been linked to a variety of biological and physiological processes, such as cell migration, proliferation, and the progression of cancer (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). FliI has been widely studied in cancer cells progression, and some of them suggest that upregulation of FliI can promote proliferation, inhibit apoptosis, and enhance cell invasion (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). On the other hand, a lower level of FliI is linked to less invasion, which slow down the growth and activation of cancer cells (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Although previous research has explored the role of FliI in cancer cells, its role in irradiated CRC remains poorly understood. The aim of the study is to determine whether FliI has a specific role in the activation and progression of irradiated FliI in the CRC cell line. We anticipate these findings to unveil the regulatory mechanism of FliI in irradiated CRC.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell lines and culture conditions\u003c/h2\u003e \u003cp\u003eThe human colorectal carcinoma cell line (HCT116) was purchased from the American Type Culture Collection (ATCC, USA) and routinely cultured in McCoy\u0026rsquo;s 5A medium (Cytiva, USA) containing 10% fetal bovine serum (FBS; Hyclone, USA) and 5% Penicillin-streptomycin solution in a humidified atmosphere of 5% CO2 at 37\u0026deg;C. When cell reached 80\u0026ndash;90% confluence, cells were detached with 5 mM trypsin/Ethylenediaminetetraacetic acid (EDTA), then re-suspended in culture media. Viable cells were counted using a hemocytometer with trypan blue staining.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eTransient siRNA transfection\u003c/h2\u003e \u003cp\u003eHCT116 cells were transfected with small interfering RNA (siRNA) targeting FliI (siFliI), using Lipofectamine\u0026trade; RNAiMAX transfection reagent (Invitrogen; ThermoFisher Scientific, Inc.), following the manufacturer\u0026rsquo;s recommendations. The cells were transfected for 48 hours for further analysis. Western Blot was done to validate this transfection. A knockdown efficiency of 70% or greater is considered effective and acceptable.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eX-ray irradiation\u003c/h2\u003e \u003cp\u003eX-ray generator (ISOVOLT 225 M2) in Division of Industrial Technology, Malaysian Nuclear Agency was set at 200kVp and 13 mA. Irradiation is made through a copper and aluminium filter with a thickness of 0.5 mm and a dose rate of 1Gy/min.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eClonogenic Assay\u003c/h2\u003e \u003cp\u003eThe cells were transfected with siRNA before X-ray irradiation with various doses (0, 2, 4, and 6 Gy) and seeded in 6-well plates. Following irradiation, cells were immediately trypsinized and plated for clonogenic survival. After 10 days, the colonies were fixed with ice cold absolute ethanol and stained with 0.1% crystal violet. Colonies of at least 50 cells were scored as survivors and counted with naked eyes or stereoscopic microscope. Plating efficiency (PE) was calculated with the following formula:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\text{P}\\text{E}= \\frac{Number of colonies counted}{Number of cells seeded} \\times 100$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eThe survival fraction (SF) was calculated with the following formula:\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\text{S}\\text{F}= \\frac{Number of colonies counted}{Number of cells seeded \\times \\left(\\text{P}\\text{E}/100\\right)}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eTo evaluate the degree of radiosensitization, the sensitizer enhancement ratio (SER) was determined by dividing the survival fraction at a 2 Gy of irradiation dose for cells without FliI-targeted transfection by the survival fraction at the 2 Gy of irradiation dose for cells with FliI-targeted transfection. Dose-response curves were graphed on a log-linear plot to illustrate the relationship with radiation dose. To model survival curves using the linear-quadratic (LQ) with an equation of \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(S=\\text{exp}\\left[-\\left(\\alpha D+{\\beta D}^{2}\\right)\\right]\\)\u003c/span\u003e\u003c/span\u003e, the Clonogenic Survival Calculation Software (CS-CAL), developed by the Translational Radiation Oncology Group at the German Cancer Research Centre, was employed. The software is accessible online at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://angiogenesis.dkfz.de/oncoexpress/software/cs-cal/\u003c/span\u003e\u003cspan address=\"http://angiogenesis.dkfz.de/oncoexpress/software/cs-cal/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eTranswell Migration and Invasion Assay\u003c/h2\u003e \u003cp\u003eTranswell migration and invasion assay were carried out using cell culture inserts with pore size of 8\u0026micro;m in 24-well plates (Corning, USA). Matrigel (Corning, USA) was diluted with serum-free medium from 8.9 mg/mL (stock solution) to 0.1 mg/mL (working concentration) and coated at the top chamber of the cell culture inserts for invasion assay. On the other hand, matrigel coating was not applied to the inserts for the transmigration assay. Approximately 5.0 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/mL of irradiated and non-irradiated transfected cells were plated in the upper chamber filled with medium containing 1% of serum. The lower chamber was filled with medium containing 20% of serum as chemoattractant. After 24h of incubation at 37\u0026deg;C, the cells were fixed in ice cold absolute ethanol and stained with crystal violet dye (Sigma-Aldrich, USA). Images were captured at twelve random fields at 200\u0026times; magnification using inverted microscope. Number of cells migrated or invaded through a porous membrane, either coated or non-coated with Matrigel were calculated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eGelatin Degradation Assay\u003c/h2\u003e \u003cp\u003eCoverslips were coated with 50 \u0026micro;g/ml of 488 Oregon Green Gelatin (Sigma-Aldrich) and irradiated and non-irradiated transfected cells were seeded on top of the coated coverslip. After 48 hours incubation, cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Cells were then stained with rhodamine-phalloidin (1:100) and Hoechst (1:1000). The coverslips were mounted on glass slides and let dried overnight at 4\u003csup\u003eo\u003c/sup\u003eC. To quantify invadopodia, 100 cells were randomly chosen from each coverslip and observed under a fluorescence microscope. The average percentage of cells displaying invadopodia was then calculated (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e).\u003cdiv id=\"Equc\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equc\" name=\"EquationSource\"\u003e\n$$\\% of cells with invadopodia=\\frac{Total number of cells with invadopodia}{Total number of cells} \\times 100$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot\u003c/h2\u003e \u003cp\u003eImmunoblotting was conducted using whole cell lysates. Irradiated and non-irradiated transfected cells were rinsed with cold PBS, lysed with RIPA buffer supplemented with protease inhibitor (Thermo Scientific, USA). Protein concentrations were determined using the Pierce\u0026trade; BCA Protein Assay Kit (Thermo Scientific, USA). Equal amounts of protein were separated by SDS-PAGE and transferred to a nitrocellulose membrane (Azure Biosystems, USA). The membranes were blocked in TBST with 5% non-fat skim milk at room temperature for 1 hour and incubated with primary antibodies overnight at 4\u0026deg;C. Subsequently, the membranes were probed with an appropriate horseradish peroxidase-linked (HRP) secondary antibody.\u003c/p\u003e \u003cp\u003eChemiluminescence was generated using Western Bright ECL HRP substrate (Advansta, USA). Antibodies against Flightless I (1:1000), GAPDH (1:5000), and HRP-linked goat anti-rabbit secondary antibodies (1:3000) were obtained from Cell Signalling Technology (MA, USA), antibodies against MMP2 (1:1000) was bought from Elabscience (Texas, USA) and antibodies against Cortactin (1:1000) was bought from Abcam (Cambridge, UK). ImageJ analysis software was used for densitometry, where intensity of the samples was standardized to the intensity of GAPDH.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eAll data presented are representative of at least three independent experiments. Results are expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM, where groups were compared by ANOVA. Statistical analysis was performed using Graph Pad Prism Version 9.5.1 software and p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered as statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSilencing the FliI Gene Increases Radiosensitivity in Irradiated HCT116 Cells\u003c/h2\u003e \u003cp\u003eClonogenic survival assays were conducted to determine whether knockdown of FliI could enhance radiosensitization in HCT116. Wild-type (WT), non-targeting siRNA (siControl) and siFliI transfected cells were exposed to various doses of ionizing radiation (0 to 6 Gy). In the absence of transfection, significant reduction in number of colonies formed were observed in all cells with increasing doses of X-ray (Fig.\u0026nbsp;1; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Interestingly, following reduced expression of FliI (siFliI), HCT116 cells become more sensitive to ionizing radiation particularly at 6 Gy (Fig.\u0026nbsp;1B; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) compared to WT and siControl of HCT116 cells. The survival fraction at 6 Gy (SF6) of siControl cells was reduced from 0.00025 to 0.00012 in the siFliI cells (Fig.\u0026nbsp;1B). The sensitiser enhancement ratio (SER) of silenced FliI compared to WT and siControl at 2, 4 and 6 Gy were ranging from 1.15 to 2.06 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0 Gy\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 Gy\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4 Gy\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6 Gy\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003esiControl\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003esiFliI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSeeding density\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e10000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 1: Effects of FliI silencing on radiosensitivity HCT116 cells by clonogenic assay.\u003c/b\u003e (A) Cell colony formation of HCT116 cell after exposed to X-ray at different doses. (B) The survival curves of WT, siControl and siFliI for HCT116 cells were plotted using the LQ model, after exposed to 0 Gy, 2 Gy, 4 Gy and 6 Gy of X-ray. Results represent mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM of three experiments with *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSensitizer Enhancement Ratio (SER) of transfected HCT116 cells (siFliI) against WT and siControl at 2, 4 and 6 Gy dose of irradiation.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSER\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWT/siFliI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003esiControl/siFliI\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSF2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.155\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.225\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSF4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.58\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSF6\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eSilencing the FliI Gene Inhibits Migration and Invasion of HCT116 Cells\u003c/h2\u003e \u003cp\u003eThe migration potential of HCT116 cells with a silenced FliI gene against irradiation was investigated using the transwell migration technique (Fig.\u0026nbsp;2A). Reduced expression of FliI gene did not significantly reduce the migratory ability of the cells (Fig.\u0026nbsp;2B; p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). However, exposure to ionizing radiation at 2, 4, and 6 Gy significantly inhibit the migratory ability of HCT116 cells. The number of migrated cells was significantly lower when FliI was knocked down and radiation was applied compared to WT and siControl (Fig.\u0026nbsp;2B; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eAdditionally, the invasive potential of FliI gene silencing cells in response to irradiation was evaluated using a transwell invasion experiment, where the extracellular matrix environment was stimulated by coating the transwell with Matrigel (Fig.\u0026nbsp;2C). Reduced FliI expression markedly impeded the invasive potential of non-irradiated cells (Fig.\u0026nbsp;2D; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Upon exposure to radiation, there was a significant reduction in the number invading HCT116 cells that were transfected with siFliI compared to non-transfected and siControl (Fig.\u0026nbsp;2D; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabb\" border=\"1\"\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0 Gy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 Gy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4 Gy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6 Gy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eB\u003c/p\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003esiControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003esiFliI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabc\" border=\"1\"\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0 Gy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 Gy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4 Gy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6 Gy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eD\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003esiControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003esiFliI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 2: FliI silencing significantly decreased migration and invasion of irradiated HCT116 cells.\u003c/b\u003e (A) Representative images of transwell migration membranes stained with 0.1% crystal violet at a magnification of 200x. (B) Graphical representative of total number of migrated cells for FliI-silenced cells compared to WT and siControl of HCT116 cells. Results represent mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM of three experiments with *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Scale bars\u0026thinsp;=\u0026thinsp;200\u0026micro;m (C) Representative images of transwell invasion membranes stained with 0.1% crystal violet at a magnification of 200x. (B) Graphical representative of total number of invaded cells for FliI-silenced cells compared to WT and siControl of HCT116 cells. Results represent mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM of three experiments with *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSilencing the FliI Gene Decreases Radiation-Induced Invadopodia-Formation in HCT116 cells.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe role of FliI expression in HCT116 cells in inhibiting invadopodia formation was investigated. Invadopodia are dynamic membrane extensions that facilitate the degradation of the extracellular matrix, contributing to cell invasion. They were detected through regions of matrix degradation, distinguished by the absence of green fluorescence in the gelatin and exhibiting co-localization with red actin spots. The blue staining (Hoechst) clearly showed the nuclei of the cells (Fig.\u0026nbsp;3A). The effect of FliI knockdown was investigated on the percentage of cells forming invadopodia in both non-irradiated and irradiated HCT116 cells. Knockdown of the FliI gene was shown to significantly inhibit invadopodia formation (Fig.\u0026nbsp;3B; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Following exposure to a 2 Gy of X-ray radiation, significant increase in the percentage of cells forming invadopodia were observed compared to non-irradiated WT, siControl, and siFliI (Fig.\u0026nbsp;3B; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Similar to non-irradiated HCT116 cells, irradiated HCT116 cells with decreased FliI expression exhibit a notable reduction in invadopodia formation, in contrast to WT and siControl (Fig.\u0026nbsp;3B; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabd\" border=\"1\"\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMerged\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFITC Gelatin\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRhodamine Phalloidin\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHoechst\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003esiControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003esiFliI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003cp\u003e+\u0026thinsp;2Gy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003esiControl\u003c/p\u003e \u003cp\u003e+\u0026thinsp;2Gy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003esiFliI\u003c/p\u003e \u003cp\u003e+\u0026thinsp;2Gy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabe\" border=\"1\"\u003e \u003ccolgroup cols=\"1\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eB\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eFliI Expression in HCT116 Cells is Upregulated with Ionizing Radiation\u003c/h2\u003e \u003cp\u003eThe effect of ionizing radiation on FliI expression was determined by Western Blot analysis (Fig.\u0026nbsp;4A). Interestingly, FliI expression increased significantly with increasing of X-ray dose (Fig.\u0026nbsp;4B; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The highest FliI expression level was observed at 6 Gy of X-ray (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabf\" border=\"1\"\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 Gy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2 Gy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4 Gy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6 Gy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFliI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"4\" morerows=\"1\" nameend=\"c6\" namest=\"c3\" rowspan=\"2\"\u003e \u003cp\u003e\u003c/p\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e145 kDa\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGAPDH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e36 kDa\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 4: FliI expression is upregulated in irradiated HCT116 cells.\u003c/b\u003e (A) Western blot analysis of FliI protein in HCT116 cells upon exposed to 0 Gy, 2 Gy, 4 Gy and 6 Gy of X-ray. (B) Graphical representation of relative expression of FliI in HCT116 cells from Western Blot analysis. Results represent mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM of three independent experiments with *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe Effects of Decreased FliI Expression and Post-Irradiation on the Protein Expression levels of Beta Catenin, Cortactin and MMP2.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe effectiveness of FliI siRNA transfection was assessed by Western Blot analysis, which revealed a significant reduction in FliI expression in HCT116 cells, resulting in estimated 80\u0026ndash;90% reduction in FliI activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003eB; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Reduced FliI expression has been shown to hinder invasion by transwell assays and gelatin degradation. Nevertheless, the specific molecular player linked to this activity has not been determined. Therefore, we selected three specific proteins, namely β-Catenin, cortactin, and MMP2, to examine their involvement in the FliI signalling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). After exposure to a radiation dose of 2 Gy, the level of MMP2 expression showed a little increase, although this change did not have statistical significance (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003eE; p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). In addition, a notable drop in FliI led to a modest reduction in β-Catenin expression in both non-irradiated and irradiated HCT116 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). However, this decrease did not reach statistical significance (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). The decrease in FliI expression did not have a significant impact on the expression levels of cortactin and MMP2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003eD and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003eE; p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabg\" border=\"1\"\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFliI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c8\" namest=\"c3\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e145 kDa\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eβ-cat\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c8\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e94 kDa\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCortactin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c8\" namest=\"c3\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e80 kDa\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMMP2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c8\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e72 kDa\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGAPDH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c8\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e36 kDa\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabh\" border=\"1\"\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB\u003c/p\u003e \u003cp\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eD\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eE\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe three cell lines used in this study (MU41, U87MG\u003c/p\u003e \u003cp\u003eand LN229) form functional invadopodia that degrade the\u003c/p\u003e \u003cp\u003eFITC-labelled gelatin which can be seen to co-localize with\u003c/p\u003e \u003cp\u003erhodamine phalloidin-stained actin puncta (Fig.1a). The\u003c/p\u003e \u003cp\u003eRadiotherapy is a very effective treatment against many types of cancer, which involves the use of high-energy radiation to target and eradicate cancer cells (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). This process disrupts the ability to divide and proliferate by inducing breaks in the DNA strands. While some cells may naturally be resistant to radiation, this could be because of DNA repair mechanisms, protective microenvironments, or genetic changes (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Despite its effectiveness, radiotherapy may have unanticipated negative effects because it paradoxically promotes the progression, migration, and invasion of cancer cells (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Exposure to ionizing radiation can trigger survival mechanisms that result in enhanced aggressiveness of cancer cells (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Numerous studies have been investigating the intricate relationship between the ionizing radiation and cytoskeletal proteins within various types of cancers, including CRC (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFliI is a cytoskeletal protein that influences tumorigenesis and cancer progression. In breast cancer cells, elevated FliI levels have been associated with increased tumor invasiveness and metastasis (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Choi et al. (2020) found that FliI acts as an inhibitor of ER-stress induced cell apoptosis in CRC cells (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). By modulating cellular Ca2\u0026thinsp;+\u0026thinsp;balance, FliI reduces the apoptotic responses triggered by ER stress. Furthermore, previous studies investigated the role of FliI in skin cancer, which regulate the cell motility and tissue invasion (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). The present study aims to investigate the relationship between FliI expression and the resistance of HCT116 CRC cells to ionizing radiation. A significant increase in FliI expression was observed in irradiated HCT116 colorectal cancer cells. Then, the expression of FliI was significantly reduced through the transfection of siRNA targeting FliI. Suppression of FliI expression was shown to enhance the radiosensitivity of HCT116 cancer cells, with the highest SER at SF6 (2.06), with the value (WT and siControl) being close to the expected value from a previous study by Chen et al. (2010) (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). This result indicates that FliI plays a role in enhancing the radio-sensitization of HCT116 cells.\u003c/p\u003e \u003cp\u003ePreviously, FliIs have been demonstrated to be involved in cell motility and tissue invasion (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). The transwell migration and invasion assay indicates that ionizing radiation alone reduced the number of migrated and invaded cells, most probably due to the reduced cell viability caused by radiation itself. However, cancer cells that could resist the effects of ionizing radiation can become more invasive. As evident from the gelatin degradation assay analysis, the data obtained in this study indicate that irradiated HCT116 cells significantly increased invadopodia formation. These results are consistent with previous studies demonstrating a significant increase in invadopodia formation when glioma and glioblastoma cells were irradiated (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Inhibition of FliI protein significantly inhibits the migration and invasion abilities of irradiated HCT116 cells. We found that reduced expression of FliI suppressed invasion by reducing the formation of invadopodia and reducing the ability of HCT116 cells to degrade gelatin.\u003c/p\u003e \u003cp\u003eStudies have shown that ionizing irradiation can enhance the invasiveness and metastatic potential of cancer cells through various mechanisms. For instance, irradiation-induced DNA damage and cellular stress responses can trigger signaling pathways involved in epithelial-mesenchymal transition (EMT), a biological process wherein epithelial cells undergo changes leading to a mesenchymal phenotype. This transition is characterized by the loss of cell adhesion and polarity, which allows cells to acquire enhanced migratory and invasive properties (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). As cells lose their epithelial characteristics, epithelial markers like E-cadherin and cytokeratins are typically downregulated, while mesenchymal markers like N-cadherin, vimentin, and fibronectin are upregulated, reflecting the acquisition of a mesenchymal phenotype (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). Numerous studies have provided evidence supporting the idea that ionizing radiation can induce changes promoting EMT in diverse cancer types. This includes instances observed in glioblastoma, lung cancer and CRC that showed a low level of epithelial markers and a high level of mesenchymal markers upon radiation exposure (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). Additionally, ionizing irradiation promotes the expression of matrix metalloproteinases (MMPs), which enhance the invasive capability of cancer cells. Studies have shown increased MMP expression in irradiated CRC and breast cancer cells (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). In this study, irradiated HCT116 cells were found to increase the invasiveness of the cells, as the percentage of cells forming invadopodia showed a significant increase. This is in line with the upregulation of FliI protein expression in irradiated HCT116 cells as a result of ionizing radiation, which enhances the invasive characteristics of HCT116 cells.\u003c/p\u003e \u003cp\u003eWhen invadopodia formation was reduced following FliI silencing, this situation led to an investigation of β-catenin, cortactin and MMP2 expression levels. β-Catenin signaling has induced metastasis in cancers such as breast cancer, hepatocellular carcinoma (HCC), and melanoma by facilitating metastatic spread through increased of cell migration and invasion (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). Furthermore, cortactin and MMP2 plays a crucial role in matrix degradation during cancer cell invasion, exhibit increased localization at invadopodia. (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). The results indicated that a significant reduction in FliI protein levels has not significantly impacted β-catenin expression, although there was a slight reduction. Previous studies have shown that when FliI is silenced, β-catenin expression reduce significantly (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). This is because of the way FliI and β-catenin are controlled. The observed discrepancy could be caused by a variety of factors, such as differences in experimental conditions, the specific cell lines used, or the degree of FliI silencing achieved. These findings also shows that cortactin and MMP2 expression remains unchanged upon FliI silencing. This suggests that the regulation of cortactin and MMP2 may be FliI-independent, thus, cortactin and MMP2 are transported and targeted via different molecular mechanisms. In addition, this independent regulation might affect other types of MMPs, such as MMP9 and MMP14, which are also involved in invadopodia formation, particularly in HCT116 cells (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). Further research is necessary to elucidate another actin cytoskeleton related to EMT, such as E-cadherin and vimentin, to determine the specific conditions under which FliI influences these pathways. Furthermore, studies have shown that inhibiting Flii suppresses CRC proliferation, migration, and invasion \u003cem\u003ein vitro\u003c/em\u003e. These findings suggest that FliI may be important in the development of new treatments that will make radiotherapy more effective and lessen the impact of aggressive metastatic CRC.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThe analysis from this study revealed a significant increase in the expression of FliI in HCT116 cells after exposure to ionizing radiation. The suppression of FliI expression resulted in increased sensitivity to radiation in HCT116 cells, along with a notable reduction in their ability to migrate and invade. Further research effort could focus on exploring alternative signalling pathways influenced by FliI, including key proteins involved in cell migration and invasion upon exposure to ionizing radiation. Additionally, assessing the influence of FliI on the epithelial-mesenchymal transition (EMT) marker post-radiation exposure could elucidate its regulatory mechanism. This investigation should use multiple colorectal cancer cell lines to ensure the results are broadly applicable and account for genetic and phenotypic variability among different cell lines, or \u003cem\u003ein vivo\u003c/em\u003e studies using animal models to validate findings in a physiological context. This result indicate that FliI may serve as a promising target for molecular therapeutics designed to regulate the aggressiveness of cancer cells thus reducing the metastasis of CRC.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eA.A.M.Y performed the experiments, analysed the data and wrote the paper. N.M.Z. and N.F.M.H conceived and designed the experiments. N.M.Z., N.F.M.H, N.A. A. and T.A.F.T.A provided intellectual inputs and analysed the data. N.E.Q.A provides optimised methodologies and guidance for various experiments. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eACKNOWLEDGEMENT\u003c/h2\u003e \u003cp\u003eThis study was financially supported by the Fundamental Research Grant Scheme (FRGS/1/2020/SKK06/UPM/02/9), Ministry of Education, Malaysia.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets used and/or analysed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBray, F. et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: a cancer journal for clinicians 74(3), 229\u0026ndash;263 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSiegel, R. L. et al. Colorectal cancer statistics, 2023. 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Cell adhesion \u0026amp; migration 13(1), 273\u0026ndash;284 (2019).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Flightless I, ionizing radiation, migration and invasion, colorectal cancer","lastPublishedDoi":"10.21203/rs.3.rs-4637992/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4637992/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Exposure to radiation is expected to inhibit the proliferation of cancer cells, but sometimes cells are resistant to ionizing radiation. This condition can lead to cancer cell metastasis and recurrence. The mechanisms leading to the development of radioresistance are not yet fully understood. Therefore, this study aims to determine the involvement of a cytoskeletal protein, Flightless I (FliI), in cancer progression and to assess the effect of radiation exposure on the role and functionality of FliI in HCT116 cells. The expression of FliI was measured in HCT116 cells, transfected with siRNA to reduce FliI activity, and validated by Western Blot. The colony formation assay revealed a significant difference in the number of cells forming colonies on FliI-silenced cells following exposure to 6 Gy radiation. Transwell migration and invasion assays shows that silencing FliI in HCT116 cells make the m less able to migrate and invade. Further investigation via a gelatin degradation assay revealed a significant reduction in the number of cells forming invadopodia in FliI-silenced HCT116 cells compared to controls. We proved the efficacy of FliI in inhibiting radiation-enhanced cancer migration and invasion, indicating its potential as a therapeutic target to enhance radiosensitization in CRC patients.","manuscriptTitle":"Flightless I as a molecular target to inhibit radiation-induced colorectal cancer metastasis.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-26 16:57:56","doi":"10.21203/rs.3.rs-4637992/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7727a1db-47c5-4235-8167-9bb91eb6ac5f","owner":[],"postedDate":"July 26th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":35012141,"name":"Biological sciences/Cancer"},{"id":35012142,"name":"Biological sciences/Cell biology"},{"id":35012143,"name":"Biological sciences/Molecular biology"},{"id":35012144,"name":"Health sciences/Medical research"},{"id":35012145,"name":"Health sciences/Oncology"}],"tags":[],"updatedAt":"2024-10-21T05:54:02+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-26 16:57:56","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4637992","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4637992","identity":"rs-4637992","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}