Background
Colorectal cancer (CRC) is the second leading cause of cancer death for men and women [
1]. Surgery is the main option for CRC therapy, and chemotherapy (CT) and/or radiotherapy (RT) could significantly decrease the local relapse and increase survival [
2]. However, many tumors resist therapies, and approximately 30% of tumors spread to distant organs, leading to poor prognosis [
3]. One of the main reasons is that currently used clinicopathological factors, even tumor stage, cannot precisely provide evidence for clinicians to design and carry out an efficient therapy strategy. Therefore, it is urgent to identify promising biomarkers for a newer approach to increase therapy response.
Ras homolog gene family member B (RhoB) proteins function as a binary switch in a wide range of signal transduction pathways [
4]. Rho GTPases are essential signal transducers in signaling pathways that regulate cell proliferation, migration, survival, and death [
5]. Rho proteins have various cellular activities and seem to play distinct, sometimes opposing roles in carcinogenesis despite having more than 85% sequence identity [
6]. RhoA is essential for contractility and proliferation via multiple growth-related gene expression pathways and actomyosin machinery. In contrast, RhoC plays a vital role in cell–cell adhesion and invasion [
7]. RhoB is involved in membrane trafficking and cell survival and has a distinctive endosome localization pattern. It has been proven that members of the Rho family, particularly RhoA and RhoC, have significantly contributed to the development of cancer [
8]. However, RhoB attracts growing interest, as its expression is altered in several cancer types. The downregulation of the RhoB in various tumor cell types has led to the hypothesis that it might function as a tumor suppressor [
6]. Recent studies have shown that RhoB expression causes apoptosis in cancerous epithelial cells and fibroblasts [
9‐
11]. DNA damage, cytokines, and growth factors might increase RhoB expression and promote oncogenesis [
12]. In parallel, other studies demonstrated that RhoB stimulates cell motility and migration or may even promote cancer metastasis [
13]. These results indicate the complex and controversial roles of RhoB in cancers. Our earlier research in a clinical trial of preoperative RT in rectal cancer patients showed that RhoB overexpression was associated with later TNM stage, distant recurrence and worse survival in the RT patients but not in non-RT patients. Our study further revealed that RhoB was related to radiation resistance through Akt-FOXM1 [
14].
In the current study, we examined the effects of RhoB together with CT on cell lines including knockout and overexpressed cell lines, zebrafish models and in tissue samples from CRC patients. This is the first study to investigate the relationship between RhoB expression, CT response and clinical outcomes in CRC patients as well as to identify the signaling pathway of the RhoB expression with CT response.
Material and methods
Cell lines
SW480 colon cancer cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA). Two SW480 knockout cell lines, SW480-KO16 and SW480-KO55, were generated at our laboratory using the predesigned RhoB-human gene knockout kit via CRISPR (#KN209837, OriGene Technologies, Rockville, MD) [
14]. The three cell lines were maintained in Eagles MEM (Sigma-Aldrich, St. Louis, MO) with 10% heat-inactivated fetal bovine serum albumin (GIBCO, Invitrogen, Paisley, UK) and 2 mM L-Glutamine at 37 °C and 5% CO2 at that temperature (Life Technologies, Carlsbad, CA). HCT116 colon cancer cell line was received from the Johns Hopkins University core cell center and kept in McCoy's 5A medium (Sigma-Aldrich) supplemented with 10% heat-inactivated fetal bovine serum albumin (GIBCO, Invitrogen, Paisley, UK) at 37 °C and 5% CO2.
Cell transfection
HCT116-OE (RhoB overexpressed) cell line was established as described below. The coding region sequence (CDS) of human
RHOB was cloned to the
pLVX-ZsGreen-PGK-Puro vector (Land Liankang Biotechnology Co., Ltd, Guangzhou, China). Cell transfection followed the manufacturer’s instructions (Land Liankang Biotechnology Co., Ltd). Cells of the negative control group were established under the same conditions. The transfection efficiency was measured through Western blot, as shown in Additional file
1: Figure S1.
Cell viability assay
Following the manufacturer's instructions, the WST-1 test (Roche, Basel, Switzerland) was used to evaluate the cell viability of the various cell lines after treatment. Briefly, 10,000 cells (SW480, SW480-KO16, SW480-KO55) or 3,000 cells (HCT116, HCT116-OE) were plated in 100 μl complete medium for 24 h before treatment and followed by incubation at 37 °C and 5% CO2. Compared to untreated or 0.1% DMSO-treated samples, the proliferation was assessed 72 h after treatment with 5-FU or OXL at increasing doses (vehicle control). Using a microplate reader, the absorbance at 440 nm was determined after adding 10 μl/well WST-1 proliferation reagent (Roche Applied Science) and incubating for 3 h.
Boyden chamber migration assay
Following the manufacturer's directions Boyden chamber migration experiments (8 m pore size, Corning, NY) were carried out. In the migration chamber, 1 × 105 cells were seeded, which were then introduced in a well plate containing complete media and incubated for 72 h at 37 °C and 5% CO2. After incubation, the cells were then fixed in 4% paraformaldehyde, stained with 0.2% crystal violet in 2% ethanol, and rinsed with phosphate-buffered saline (PBS). A light microscope was used to acquire pictures of moving cells (Zeiss Lab.A1, Jena, Germany). Under an x-100 magnifying lens, the migration rate was evaluated by counting the number of cells that passed through the filter.
Detection of reactive oxygen species (ROS)
By using the manufacturer's recommended reactive oxygen species assay kit, intracellular ROS levels were measured (BioVision, Inc., Milpitas, CA). Cells were retrieved and reconstituted in 100 μM dihydro-dichlorofluorescein diacetate (H2DCFDA) with serum-free media after a 72-h treatment with 5-FU and OXL. Intracellular H2DCFDA was esterified to dichlorodihydrofluorescein, which ROS then oxidized to create the fluorescent substance dichlorofluorescein. The expression level was assessed by measuring fluorescence using plate reader after a 45-min incubation period at 37 °C (Excitation 495 nm, Emission 529 nm).
Caspase 3 activity measurement
Caspase 3 activity in SW480, SW480-KO55, SW480-KO16, HCT116 and HCT116-OE cell lines were estimated using a caspase 3 activity assay kit, according to the manufacturer's protocol (BioVision, Inc.). The cells were briefly lysed in a buffer solution containing a caspase 3 sample (BioVision, Inc.). The homogenates of cultured cells were then clarified by differential centrifugation at 10,000 × g, and 4 °C for 10 min and the supernatant was collected. The cell lysates (200 µg) were then introduced to the DEVD substrate conjugate for 2 h at 37 °C followed by supernatant collection. The samples were measured in a microplate reader at an excitation of 405 nm.
Autophagy microplate assay
The autophagy flux was determined by using a Cyto-ID autophagy detection kit following the instructions provided by the manufacturer (Enzo, UK). Briefly, 10,000 cells (SW480, SW480-KO16, SW480-KO55) or 3,000 cells (HCT116, HCT116-OE) were seeded and incubated for 24 h in a 96-well plate at 37 °C. Chloroquine (10 μM), Rapamycin (0.5 μM), OXL, and 5-FU (IC
50 concentrations, Table
1), and negative control were added. The medium was cautiously taken out and discarded after treatment. A dual-color detection solution was applied to each well (100 μL) after cells had been rinsed with 1X Assay Buffer (100 μL). For 30 min at 37 °C, the plate was incubated in the dark. To eliminate excess dye, a new 1X Assay Buffer (100 μL) was added to each well, before which the cells were washed twice with 1X Assay Buffer (200 μL). The Hoechst 33342 Nuclear Stain was assessed with a DAPI filter (Excitation 480 nm, Emission 530 nm), and the CYTO-ID Green detection reagent was measured with a FITC filter (Excitation 340 nm, Emission 480 nm).
Table 1
Cell lines treated with 5-flourouracil (5-FU) and oxaliplatin (OXL) at different concentrations (0–60 μM) for 72 h at 37 °C. IC50 values correspond to the concentration required to reduce cell growth by 50% compared to control cells. DMSO was used as positive control
SW480 | 38.5 ± 0.8 | 21.9 ± 0.4 |
SW480-KO16 | 27.2 ± 0.5** | 17.8 ± 0.3 |
SW480-KO55 | 25.9 ± 0.4** | 18.8 ± 0.9 |
HCT116 | 51.2 ± 0.3 | 30.4 ± 0.5 |
HCT116-OE | 54.8 ± 1.1 | 35.7 ± 0.7 |
RNA-seq library generation and sequencing
RNA was extracted using phenol–chloroform method, utilizing kits manufactured by BGI (Shenzhen, China). Nearly 10–50 mg of tissue samples were powdered using liquid nitrogen and transferred into a tube containing 1.5 ml of Trizol reagent. The homogenized samples were centrifuged at 12000xg for 5 min at 4 °C.
The supernatant was transferred to a new tube and mixed with 1.5 ml of Trizol reagent along with 0.3 ml of chloroform/isoamyl alcohol (24:1). For 15 s, the tubes were given a vigorous shake followed by 10 min of centrifugation at 12000 g at 4 °C. The aqueous phase was transferred to a new tube where an equal amount of isopropyl alcohol was added. Further, the tubes were centrifuged at 19,645 g for 20 min at 4 °C. After the removal of the supernatant, 1 ml of 75% ethanol was used to wash the RNA pellet. Later, the air-dried pellet was treated with 25–100 μl of DEPC water depending on the RNA concentration. The RNA library was prepared as per BGI, China’s protocol. Thereafter, Qubit 3.0 Fluorimeter and Agilent 2100 Bioanalyzer were used to quantify the purified libraries. And further sequencing was performed.
RNA-seq alignment and quality control
Raw reads containing more than 50% of low-quality bases, defined as bases with a sequence quality of no higher than 5% or adapters read with more than 10% unknown bases, were removed to minimize data noise. After quality control, clean reads were mapped to the reference transcriptome using Bowtie2 [
15] with default parameters (performed by BGI, China). The quantification tool for RNA-Seq by Expectation–Maximization was used to compute maximum likelihood abundance estimates for transcripts that were isoforms of the same gene. The fragments per kilobase of exon per million fragments (FPKM) values were calculated according to the standard protocol provided by BGI, China. Differential expression analysis was carried out using a linear method within the Limma R package [
16] with Benjamini–Hochberg FDR for multiple hypothesis testing. Genes with a fold change greater than 1 and adjusted p-value below 0.05 were considered statistically significant. A summary of the genome mapping for samples can be found in Additional file
2: Table S1.
Gene-set enrichment analysis
We tested the gene ontology (GO) terms and pathways for over-representation in the set of genes that had either significant differential gene expression or at least one significantly differential splicing isoform. We separately tested up- and down-regulated genes. GO was applied to annotate meaningful gene products of biological processes (BP). And Kyoto Encyclopedia of Genes and Genomes (KEGG) were utilized to identify the target genes in biological pathways. We used the cluster Profiler R package [
17] to analyze the GO terms and KEGG pathways. Statistical significance was obtained using the Benjamini–Hochberg FDR method (p < 0.05).
Zebrafish
At the zebrafish facility at Linköping University, transgenic Tg (fli1: EGFP) zebrafish (ZIRC, Eugene, OR) were kept in accordance with standard organizational procedures. Zebrafish embryos were produced by natural mating, and the following morning, post-spawning, the eggs were collected. The eggs were then cleaned, examined for evidence of successful fertilization, and incubated for injection using an E3 medium enriched with 1-phenyl-2-thiourea at 28.5 °C in humidified ambient air.
The cell lines were cultured at 37 °C and 5% CO2 for 24 h after being labeled with 1,1'-dioctadecyl 3,3,3′3'-tetramethylindocarbocyanine (DiI) at a concentration of 5 ng/ml in PBS for 1.5 h. Cells were gathered and introduced into the perivitelline region of 48-h-old embryos after being labeled. The embryos containing tagged cells in the bloodstream were excised after injection. The number of cells injected was the same across all treatment groups and barely varied between embryos. At 28.5 °C, the embryos (20 per group) were incubated in humidified room air. After 24 h, the embryos were given 0.004 percent tricaine anesthesia to observe the cells under a fluorescent microscope (Nikon D-eclipse C1, Tokyo, Japan).
Patients
The study included 260 patients from the Southeast Health Care Region of Sweden who were diagnosed with CRC between 1984 and 2013, and the detailed information of the patients has been described at the Additional file
2: Table S2.
The RhoB expression was determined by immunohistochemistry in 189 surgical samples of primary tumors, 72 samples of regional lymph node metastases, 143 samples of normal mucosa adjacent to the tumor tissue (histologically free from cancer and taken from the margin of distant surgical resection) and 203 samples of normal mucosa distant to the tumor tissue (histologically free from adjacent between cancer and normal mucosa. The 4 μM tissue microarray slides from paraffin-embedded blocks were deparaffinized in Aqua de Par 10 Ancillary reagent (ADP1002 M, BioCare Medical) for about 20 min. After being kept at 65 °C for 2 h, all sections were then placed into a pressure cooker Decloaking Chamber NexGen configured in a temperature cycle to achieve a maximum of 110 °C for 5 min to perform heat-induced epitope retrieval using Borg Decloaker RTU antigen retrieval solution (BD1000). After cooling, the slides were rinsed in tap water before being washed in tris-buffered saline (TBS). The slides had been washed in TBS before incubating with a rabbit polyclonal primary anti-RhoB antibody (Santa Cruz, catalog no: sc-180) for 30 min at room temperature while being blocked for 5 min by peroxidase. The tertiary antibody MACH4 Universal HRP-Polymer and the secondary antibody MACH4 Universal HRP-Probe were then applied to the slides for 10 min (BRR 4012). TBS was used to wash the slides after cooling down and then rinsed in the gentle flow of running water. The slides were rinsed in TBS and then treated with a peroxidase inhibitor for 5 min to prevent endogenous peroxidase. Following the detection of HRP, the slides were subsequently rinsed with TBS wash buffer. The slides were coated with diaminobenzidine (DAB) chromogen and 1.0 ml of DAB substrate buffer before being incubated for 5 min. The slides were counterstained with Mayer’s hematoxylin staining solution and mounted with PERTEX mounting medium after being rinsed in deionized water.
The immunostaining was evaluated independently by two investigators (MK and CH) without knowledge of clinicopathological data. Staining intensity in or tumor cells was graded according to the following criteria: Immunostaining was evaluated by a semi-quantitative method according to the percentage of positive normal epithelial cells or tumor cells: 0 (negative); 1 (1–25%); 2 (26–50%); and 3 (> 50%). In the case of discrepancy, the sections were re-examined, and a consensus score was reached. Cases with scores of 2 or less were classified statistically as low-expressing groups, whereas those of 3 or more were classified as high-expressing groups.
Molecular docking analysis of RhoB
Molecular interaction of RhoB with chemotherapy drugs
To study the association of RhoB to the chemotherapy response, it is essential to understand the possible molecular interaction between the RhoB protein and the chemotherapeutic drugs through molecular docking analysis. For the molecular docking analysis of the RhoB protein and the chemotherapy drugs, protein, ligand, and grid optimization were prepared using AutoDock Tools 4.2 before initiating the molecular docking analysis [
18]. The RhoB protein's x-ray crystallographic 3-dimensional (3-D) structure (PDB id: 2FV8) was retrieved from the protein data bank (PDB) database. The PubChem database was used to obtain the structures of chemotherapy drugs 5-FU (CID: 3385) and oxaliplatin (CID: 9887053). The 3-D structure of 5-FU in SVG format was converted to PDB format during ligand preparation. Since the 3-D structure of OXL is unavailable, the 3-D structure in PDB format was generated using the canonical SMILES. Afterward, the bonds were made rotatable using the torsion tree, optimized, and converted to PDBQT format. The RhoB was then prepared by removing the water molecules, heteroatoms, and co-crystallized solvents. Then, polar hydrogen atoms and Kollman partial charges were added to the structure. The grid box comprising the coordinates x, y, and z, mapping, and size parameter was determined, and molecular docking between the protein and ligands was performed using AutoDock Vina [
19]. The results were then analyzed in the Discovery Studio visualization tool [
20]. The active binding site amino acid sequences interacting with the chemotherapy drugs were analyzed in the InterPro web server to identify the drug interaction with the binding domain of the protein.
Interaction analysis of RhoB with Caspase 3 protein
RhoB and caspase 3 docking was performed on the protein–protein docking web server to identify the molecular interaction in regulating RhoB-mediated caspase 3 expression. Similar to the protein preparation of RhoB, the 3-D structure of caspase 3 (PDB id: 1CP3) was retrieved from the PDB database. Before docking, the water molecules, heteroatoms, protein-bound miRNA, and ligand were removed. The protein–protein docking analysis was performed with RhoB and caspase 3proteins using High Ambiguity Driven Biomolecular Docking (HADDOCK) webserver v2.4 [
21,
22]. The HADDOCK will cluster the docked protein structures and the best-docked structure of the topmost cluster based on the lowest HADDOCK score. The molecular interaction between the protein complexes was further analyzed in the PRODIGY webserver [
23,
24] and visualized using the PyMOL tool. The 2-D interaction map was generated to identify the interacting amino acids and their type of interaction using LigPlot [
25].
Statistical analysis
All the experiments of cell lines and zebrafish studies were performed with minimum triplicates, if not specified otherwise and presented as mean ± standard deviation or standard error of the mean. Statistical significance was determined using two-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. P < 0.05 were considered statistically significant. Statistical analyses and graphs were made using GraphPad Prism 9. To test differences in RhoB expression levels in primary tumors, adjacent normal mucosa, distant normal mucosa and metastases, the McNemar's test was used. The Chi-squared test was used to compare the clinicopathological factors. The log-rank test was used for the survival analyses, and survival curves were calculated using the Kaplan–Meier method. The outcome for the survival analyses was 10 years OS. Adjusted Hazard ratio (HR) and 95% confidence interval (CI) were calculated using multivariate Cox proportional hazard model. All statistical analyses for the clinical part were performed using Statistica software, with p < 0.05 considered statistically significant.
Discussion
The present study has demonstrated that high RhoB expression in patients with stage III tumors that received chemotherapy had worse survival compared to lower RhoB expression; there was no such association in patients who did not receive chemotherapy. The evidence was further validated in zebrafish and cell models. We observe that RhoB knockout cell lines exhibited increased sensitivity to the drug treatments concerning cellular proliferation and apoptosis. Furthermore, gene set enrichment analysis revealed that the differentially expressed genes in the RhoB-treated group were predominantly enriched in the signaling pathways related to cellular proliferation, migration, and p53, compared to the control group.
Our previous study revealed an independent association between RhoB overexpression and poor survival in rectal cancer patients who underwent radiotherapy. In RhoB knockout cells, the expression of FOXM1 was reduced. Downregulation of FOXM1 occurred in RhoB knockout cells, contributing to reduced survival rates and diminished migration and invasion capabilities following radiation. Among patients undergoing radiation therapy, RhoB overexpression correlated with elevated FOXM1 levels, advanced tumor, local and distant metastasis, and independently predicted poor survival, irrespective of other clinical factors [
14]. Interestingly, the current findings demonstrate that RhoB overexpression was also associated with worse survival in patients who received chemotherapy. Conclusively, the expression of RhoB significantly impacts the response of CRC patients to either radiotherapy or chemotherapy, suggesting that RhoB provides new insights into improving therapeutic interventions for CRC.
The molecular docking results suggested that the OXL had a better affinity with a maximum of 8 amino acid residues interacting with the ligand. Though OXL had better binding energy, 5-FU also had a negative binding energy with 3 hydrogen bonds and 2 hydrophobic interactions with RhoB. The molecular interaction of OXL with RhoB with the least binding energy added weightage to the strong association between the RhoB protein and the chemotherapeutic drug. The binding of OXL to the pocket region of RhoB had maximum interactions with the amino acid residues compared to the 5-FU suggesting that the OXL might be the key interactor in regulating the RhoB expression and its function. In coherence, the docking analysis of the RhoB protein with 5-FU and OXL showed that modulating the expression of RhoB with OXL might inhibit the molecular interaction between the RhoB and caspase 3 protein to induce apoptosis in tumor cells. Moreover, most of the interacting amino acids of RhoB with 5-FU and OXL were classified in the small GTP binding domain, indicating that the chemotherapy drugs might inhibit the function of RhoB regulating cytoskeletal reorganization, cell polarity, cell cycle progression, and gene expression in a cell. In elucidating the regulation of apoptotic cascade with the 5-FU and OXL through the RhoB-mediated response, it is essential to delineate the molecular interaction between the RhoB and caspase 3 proteins. RhoB-caspase 3 complexes showed a higher structural and electrodynamic stability indicating that the RhoB might involve in the negative regulation of apoptosis. The increase in the caspase 3 activity on RhoB knockout cells provided additional weightage. HADDOCK results predicted the molecular interactions between the RhoB and capsase3 proteins. The hydrogen bonds between the RhoB and caspase3 amino acid residues indicate a stronger binding affinity. In addition, the maximum number of hydrophobic interactions provides better stability and dynamics to the protein complexes. Further, the HADDOCK analysis in the present study has shown that RhoB had a strong association with the mitochondrial-mediated apoptotic regulatory caspase 3 proteins.
The influence of drug-induced ROS on apoptotic cell death has been utilized extensively in developing effective cancer chemotherapy strategies. Numerous studies examining the effect of anticancer agents on CRCs have already demonstrated that most of these compounds influence ROS [
26]. According to our findings, 5-FU and OXL probably caused ROS production in the cells, and RhoB may be involved in the stress response process. However, this pathway is yet to be fully understood; this might be the primary mode of action for many drugs used in anti-cancer therapy. It is still unclear how cellular apoptosis affects the clinical outcome in CRCs. To delve deeper into the involvement of RhoB in chemotherapy-induced cell death, we examined the autophagy flux in cells following treatment with 5-FU and OXL. This evaluation validated the correlation between RhoB and caspase 3 activity in cells subjected to chemotherapeutic drug treatment. The outcomes showed that RhoB positively impacts autophagic flux in the SW480, SW40KO16, and SW480-KO55 cell lines following 5-FU and OXL treatment. Moreover, the SW480-bearing zebrafish tended to develop a larger tumor than the SW480-KO-bearing zebrafish after being injected with SW480 and SW480-KO cells. As a result, the findings showed that RhoB was involved in caspase-3-dependent apoptosis in the cells after treatment with 5-FU and OXL.
To further investigate the involvement of RhoB at the transcriptomic level, we conducted RNA-seq analysis on HCT116-OE, HCT116-WT, SW480-KO and SW480-WT cells treated with 5-Fu and OXL, respectively. We observed numerous upregulated and downregulated gene expression levels across the groups (Additional file
3: Table S3). We further carried out GO and KEGG pathway enrichment analysis for the groups based on the DEGs. Our results demonstrated that SW480 vs. SW480-KO16 and HCT116 vs. HCT116-OE1, had comparable biological functions, cellular components, and different signaling pathways. Most genes essential for cellular processes were down-regulated in the SW480 vs. SW480-KO16 group, whereas most genes are up-regulated in the HCT116 vs. HCT116-OE1 group. In coherence, our findings suggest that RhoB was crucial for all tested cell lines to migrate after 5-FU and OXL treatment. In addition, the MAPK signaling pathway and cell adhesion molecules were the most representative KEGG pathways associated with cancer for the SW480 group in comparison to SW480-KO16. Whereas most genes were up-regulated in the HCT116 vs. HCT116-OE1 group. In coherence, our findings suggest that RhoB is crucial for all tested cell lines to migrate after 5-FU and OXL treatment. However, signaling pathways like MAPK, TNF, Ras, and PI3K-Akt emerged as the most prominent KEGG pathways associated with the comparison between HCT116 and HCT116-OE1. Thus, caspase-3 activity results showed that RhoB was involved in caspase-3-dependent apoptotic cell death and positively influenced autophagic flux in 5-FU and OXL-treated SW480, SW40KO16, and SW480-KO55 cell lines. Further, we counted the RhoB OE up-regulated genes and KO down-regulated genes in the 5-FU-treated group and OXL treated group, respectively. In the 5-FU treated group, there were 20 OE up-regulated genes that overlapped with KO down-regulated genes. Similarly, 17 OE up-regulated genes overlapped with KO down-regulated genes in OXL treated group (Additional file
4: Table S8). We merged these two overlapping gene sets (Total 31 genes) to perform GO biological process and KEGG enrichment analysis. Many pathways related to cell proliferation and growth as well as hair development were found (Additional file
5: Table S9, Additional file
1: Figure S7). This is consistent with the reported role of RhoB in regulating cell proliferation, growth, and transformation [
27]. Adly et al
. [
28] found that RhoB protein was strongly expressed in the various elements of the human scalp skin and hair follicles [
28]. That was also consistent with our results.
Our comprehensive analysis reveals that RhoB plays a pivotal role in modulating the response of CRC cells to chemotherapy. While RhoB overexpression in HCT116-OE cells demonstrated resistance to 5-FU and OXL, the intricate relationship between RhoB, ROS production, and caspase-3 activity underscores the complexity of its involvement in chemotherapy response. Moreover, to assess the impact of RhoB on the cellular response, RhoB knockout cell lines (RhoB KO 16 and RhoB KO 55) were generated from SW480 cells through the implementation of the CRISPR/Cas9 system and the protein expression of RhoB was shown in Additional file
1: Figure S1. The successful establishment of these knockout cell lines was confirmed by the complete depletion of RhoB expression. Notably, this genetic manipulation did not induce significant alterations in the expression levels of closely related GTPases, RhoA and RhoC, underscoring the specificity of RhoB knockout in this experimental model [
14].
Reviewing the results of previous studies on the role of RhoB in cancers, RhoB has been implicated in both oncogenic and tumor suppressor functions, highlighting its complex involvement in cancer development and progression. In some studies, RhoB exhibits oncogenic properties by promoting cell proliferation and growth, and reducing therapy response, resulting in poor clinical outcomes including primary tumor metastasis and short survival of patients including colorectal cancers [
14,
29,
30]. On the other hand, RhoB has demonstrated tumor suppressor characteristics by inhibiting cell invasion and metastasis, and enhancing therapy response, leading to better clinical outcomes such as bladder, ovarian and head-neck cancers [
31‐
34]. These controversial results may arise from variations in tumor types, animal and cell models, along with the diverse treatments and techniques employed in the different studies. Besides, comprehending the dualistic nature of RhoB in cancers necessitates elucidating the biological factors that govern its behavior through different signaling pathways not only in tumor cells but also tumor cell microenvironment.
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