RHOJ enhances adhesion and proliferation capabilities and suppresses apoptosis of melanoma cells by activating the Rap1 signaling pathway

Introduction

Skin cancer poses a significant risk to life, with melanoma being the most dangerous form, responsible for 80% of all skin cancer-related fatalities. Melanoma arises from the malignant transformation of melanocytes, the cells that produce melanin, a pigment that protects the skin from harmful ultraviolet radiation (1,2). Over the past decade, there have been unprecedented clinical advancements in the treatment of advanced or metastatic melanoma, with a wide range of new therapies introduced, including immunotherapy, targeted therapy, and their combinations (3,4). In melanoma research, there are still gaps in understanding the mechanisms of adhesion and metastasis. Current studies often do not fully elucidate the specific roles of these mechanisms in melanoma progression or identify their potential therapeutic targets (5,6).

RHO GTPases, a diverse family of small signaling proteins, play a pivotal role in regulating cell membrane dynamics and various critical cellular processes. Among them, RHOJ is a notable member that contributes to the intricate network of cellular functions. These proteins are essential for controlling the cell cytoskeleton, which directly impacts cell morphology and polarity. They orchestrate changes in cell shape, enabling cells to adapt to different environments and perform essential functions such as migration and adhesion (7). They govern a variety of cellular functions essential for managing the cell’s internal framework, including the regulation of cell shape and orientation, cellular movement, vesicular trafficking, and cytoplasmic division (8). Furthermore, the RHO family of GTPases plays a crucial role in the dynamics of cellular movement, facilitating cell migration during processes such as growth, tissue repair, and the body’s defense mechanisms. They also play a significant role in vesicular transport, which is essential for the proper distribution of cellular materials and communication between different cellular compartments (9). In melanoma, RHOJ can influence tumor progression and metastasis by affecting how cancer cells migrate and interact with their environment. These findings highlight the complex regulatory network controlled by RHOJ in melanoma, suggesting that targeting these pathways could provide new therapeutic strategies to inhibit tumor progression and improve treatment outcomes (10). RHOJ’s involvement suggests that disrupting its regulatory mechanisms could be effective in limiting melanoma growth and metastasis (11).

Additionally, small RAP1, a key player in the signaling cascades that activate integrins within the cell, is also significant. RAP1’s role in promoting integrin activation is crucial for cell adhesion and migration (12). Dysfunction in RAP1 signaling is closely associated with increased tumor metastasis and invasion, highlighting its potential as a therapeutic target. RAP1 regulates cell adhesion and motility by interacting with integrins and other adhesion molecules. In melanoma, altered RAP1 signaling can enhance cell migration and invasion, contributing to metastasis. Targeting RAP1 signaling pathways may offer new strategies for controlling tumor spread and improving patient prognosis (13).

Therefore, we aim to explore the roles of RHOJ and Rap1 signaling pathways in regulating melanoma cell adhesion, proliferation, and apoptosis at the cellular level. Understanding the mechanisms of these pathways could provide crucial insights for developing targeted therapies to manage melanoma more effectively. We present this article in accordance with the MDAR reporting checklist (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2024-2692-b/rc).

Methods

Materials

This study utilized various human melanocyte cell lines for experimental analysis. The normal melanocyte cell line PIG1 (RRID:CVCL_S410. CRL-4059, ATCC, Manassas, VA, USA) exhibited stable growth characteristics and served as a control group for comparative experiments. Melanoma cell lines included the highly invasive and metastatic A375 cells (RRID:CVCL_0132. CRL-1619, ATCC, USA), the atypical subtype A2058 cells (RRID:CVCL_1059. CRL-3601, ATCC, USA) with specific genetic and phenotypic features, and the HS294T cells (RRID:CVCL_0331. HTB-140, ATCC, USA) demonstrating distinct biological behavior suitable for detailed analysis and validation experiments. The cells were cultured at 37 ℃, and maintained in complete Mφ medium (GIBCO, Grand Island, NY, USA) and 5% CO₂. Additionally, the study employed the chemical activator 8-pCPT-2'-O-Me-cAMP (Sigma-Aldrich, St. Louis, MO, USA), validated in literature (14), known for effectively activating Rap1 protein activity.

Gene Expression Omnibus (GEO) data analysis and bioinformatics analysis

The research utilized the Gene Expression Profiling Interactive Analysis (GEPIA) database (accessible at http://gepia2.cancer-pku.cn/#index) to conduct a series of examinations, which included analyzing 461 melanoma tissue samples and 558 samples of normal melanocyte tissue. The study aimed to contrast the expression levels of the RHOJ gene between melanoma and normal melanocyte tissues, employing the GEPIA analytical tools specifically designed for RHOJ gene expression examination. Additionally, using the GSE122907 dataset (https://metadataplus.biothings.io/geo/GSE122907), it analyzed the expression patterns and differences of the RHOJ gene between melanoma tissues and normal melanocyte cells, conducting GEO dataset analysis. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments.

RNA extraction and real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR)

RNA isolation was performed utilizing a specialized RNA isolation system (Qiagen product code 74104, produced in Hilden, Germany). Subsequently, complementary DNA synthesis was achieved through the use of a reverse transcription reagent set (Qiagen, item number 205311, origin: Germany), adhering to the producer’s instructions. For the polymerase chain reaction, the SYBR Green qPCR mix (Qiagen, reference number 204143, Germany) was employed. Following the conclusion of the polymerase chain reaction (PCR) processes, the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was employed as a standard for mRNA quantification, and the quantitative analysis of relative expression was computed via the 2^−ΔΔCT quantitative method (15).

Western blot analysis

The cells affected by melanoma were disrupted utilizing a radioimmunoprecipitation assay (RIPA) buffer solution (Bio-Rad product, item code 170-8171, origin: Hercules, CA, USA). Post disruption, the resultant supernatants were normalized to a uniform protein concentration, assessed via the Pierce™ BCA Protein Quantification Kit (Thermo Fisher Scientific, part number 23225, Waltham, MA, USA). These lysate specimens were subsequently combined with a lysis solution (Thermo Fisher Scientific, item code 9803, USA) and augmented with a 5% concentration of loading buffer (Sigma-Aldrich, reference number S3401, USA) before being subjected to heat treatment for a duration of 10 minutes at a temperature of 100 ℃. The proteins were afterward resolved using 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with Bio-Rad Mini-PROTEAN^® TGX™ Gels (product number 456-1094, USA), running at a steady voltage of 80 V for a total of 90 minutes. Post-electrophoretic separation, the proteins were blotted onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad Trans-Blot^® Turbo PVDF Membranes, item code 1704158, USA). To inhibit non-specific bindings, the membranes were treated with a 5% solution of non-fat milk in Tris Buffered Saline with Tween-20 (TBST) buffer (Thermo Fisher Scientific - 1X TBS-T Buffer, catalog number 28376, USA) for 2 hours at ambient temperature while agitating. An antimicrobial compound, PVDF-Ag, was applied to the PVDF membrane and left to act at 4 ℃ for an entire night. Following this, the membranes were exposed to polyclonal antibodies targeting RHOJ (product code PA5-48271, diluted 1:1,000, Thermo Fisher Scientific, USA) and GAPDH (product code ab9485, diluted 1:2,000, Abcam, Cambridge, UK), and finally treated with a Goat Anti-Rabbit immunoglobulin G (IgG) H&L secondary antibody conjugated to horseradish peroxidase (HRP) (product number A0208, diluted 1:5,000, Beyotime, Beijing, China). Blots were evaluated by enhanced chemiluminescence. GAPDH as an internal reference protein. The expression of the above protein levels was quantified densitometrically using the ImageJ (V 15, NIH, Bethesda, MD, USA) (16).

5-ethynyl-2'-deoxyuridine (EdU) detected cell proliferation

A375 cells were seeded at a concentration of 2×10⁴ cells/ml during their logarithmic phase and counted using standard techniques. Each well of a 96-well plate was filled with 200 µL of this cell suspension and incubated at 37 ℃ with 5% CO₂ for 8–10 hours to promote adherence. The medium was subsequently replaced, and the cells were cultured for an additional 48 hours. The EdU solution (C10310, Beyotime, Beijing, China) was diluted 1,000-fold in F1 medium (Thermo Fisher Scientific, catalog number 12100046, USA) containing 10% fetal bovine serum (FBS) (Gibco, catalog number 26140079, USA) to achieve a final concentration of 50 µM. Each well received 100 µL of this 50 µM EdU medium and was incubated at 37 ℃ with 5% CO₂ for 2 hours. Following incubation, cells were washed twice with phosphate buffer saline (PBS), fixed with 50 µL of 0.2% glycine (Alfa Aesar, catalog number A17822, Ward Hill, MA, USA) in PBS for 30 minutes, and then washed again. The cells were permeabilized using 100 µL of permeabilization buffer (Abcam, catalog number ab183917, UK) for 10 minutes, followed by another PBS wash. Then, 100 µL of 1× Apollo staining solution (Beyotime, catalog number C1039, China) was added to each well and incubated in the dark for 30 minutes. After removing the staining solution and washing with permeabilization buffer, 100 µL of methanol (Sigma-Aldrich, catalog number 322415, USA) was added for 5 minutes, followed by a PBS wash. For nuclear staining, 100 µL of 1× Hoechst 33342 reaction solution (Thermo Fisher Scientific, catalog number H3570, USA) was applied to each well and incubated in the dark for 30 minutes. The staining solution was removed, wells were washed with PBS, and finally, 100 µL of PBS was added for storage until examination under a fluorescence microscope (17).

Flow cytometry analysis of cell proliferation in different groups

After reaching confluence at approximately 7×10⁵ cells per mL in the culture flask, serum-free F12 medium (Thermo Fisher Scientific, catalog number 11320033, USA) was substituted and cells were synchronized for 24 hours. After implementing the specified interventions for each group, cells were harvested, centrifuged at 1,000 rpm for 5 minutes, and the supernatant was removed. Following this, 2 mL of PBS was added and mixed with the cell pellet. The cells were then centrifuged again at 1,000 rpm for 5 minutes, with the supernatant discarded once more, and the cells were resuspended. Flow cytometry (BD FACSymphony™ A5, San Jose, CA, USA) was subsequently carried out according to the cell cycle kit’s protocol to assess the distribution of cells across various phases of the cell cycle (18).

Cell adhesion test with cell adhesion detection kit

The adherence evaluation of melanoma A375 cell line was conducted utilizing a specialized cell adherence kit (purchased from Sigma-Aldrich, item code C9472, origin: USA). To commence, the cells were seeded into 96-well culture dishes at a seeding density of 10,000 cells per individual well and left to attach for a duration of 60 minutes at a temperature of 37 ℃ within a moisture-rich atmosphere containing 5% carbon dioxide. Following the attachment phase, cells that had not adhered were gently rinsed off with phosphate-buffered saline. The cells that remained adhered were then preserved with 4% paraformaldehyde (sourced from Bio-Rad, product number 161-2407, manufactured in the USA) for a quarter of an hour at ambient temperature before being dyed with a crystal violet staining solution (procured from Sigma-Aldrich, product ID C0775, from the USA) for a 10-minute interval. Any surplus dye was discarded through PBS rinsing, and the plates were left to dry at room temperature. For the quantification of cellular adherence, the crystal violet stain that had bound to the cells was dissolved in a 10% solution of acetic acid (acquired from Sigma-Aldrich, item code A6283, USA), and the absorbance was recorded at a wavelength of 595 nm using a microplate photometer (model PHERAstar FSX by BMG LABTECH, made in Ortenberg, Germany). All experiments were conducted in triplicate to ascertain the uniformity and statistical significance of the findings.

Assessing the impact of RHOJ knockdown on Melanoma cell viability using Cell Counting Kit-8 (CCK-8)

Melanoma cells were cultured at 37 ℃ in a 5% CO₂ atmosphere using Dulbecco’s Modified Eagle Medium (DMEM) medium (Thermo Fisher Scientific, catalog number 11995065, USA), supplemented with 10% FBS and 1% penicillin/streptomycin (Sigma-Aldrich, catalog number P0781, USA). Once the cells reached approximately 80% confluence, they were transferred to 96-well plates, with around 5,000 cells per well. To achieve RHOJ gene knockdown, siRNA RHOJ (Qiagen, Germany) was introduced into A375 cells using suitable transfection reagents (Invitrogen, Lipofectamine™ 2000, catalog number 11668027, Carlsbad, CA, USA), following the manufacturer’s protocol. Sequences for RHOJ siRNA (5'-UUUCAGCAGGCUCACAUCCAG-3') were designed and confirmed, with si-Ctrl used as a negative control. After 12, 24, and 48 hours of incubation post-RHOJ knockdown, the cells were assessed for activity using the CCK-8 reagent (Dojindo Molecular Technologies, catalog number CK04, Japan). Cells at each time point were processed according to the manufacturer’s guidelines, and absorbance [optical density (OD) value] was measured to evaluate the cells’ metabolic activity.

Immunofluorescence detection

During the study, the technique of immunofluorescence was deployed to pinpoint the adhesive proteins that play a role in both intercellular and cell-to-matrix connections. The cells were seeded onto glass coverslips placed in 24-well culture dishes at a seeding density of 100,000 cells per well and left to adhere for an entire night at a temperature of 37 ℃ within an atmosphere of 5% carbon dioxide. The subsequent morning, the cells underwent fixation with a 4% solution of paraformaldehyde (produced by Bio-Rad, with the catalog number 161-2407, originating from the USA) for a quarter of an hour at ambient temperature. They were then made permeable by the application of 0.1% Triton X-100 (distributed by Thermo Fisher Scientific, identified by catalog number T8787, USA) diluted in PBS for a duration of 10 minutes. Post permeabilization, the cells were blocked with a 3% solution of bovine serum albumin (BSA) (also from Bio-Rad, catalog number 170-6404, USA) in PBS for a period of one hour to reduce any unspecific binding. The primary antibodies, aimed at proteins involved in cell adhesion such as E-cadherin (at a dilution of 1:100, catalog number ab76055) and vinculin (at a dilution of 1:200, catalog number ab18058), both from Abcam, Cambridge, USA, were prepared according to the manufacturer’s instructions and allowed to interact with the cells for an extended period of time, specifically overnight at 4 ℃. After rinsing with PBS, the cells were exposed to secondary antibodies conjugated with fluorophores [Anti-mouse IgG H&L (Alexa Fluor^® 488, Eugene, OR, USA), diluted 1:1,000, catalog number ab150077, Abcam, USA] for an hour at room temperature, shielded from light. Following a further PBS rinse, the coverslips were mounted onto microscope slides using a medium that included DAPI (supplied by Thermo Fisher Scientific, product number D1306, USA) to stain the cell nuclei. The fluorescent imagery was captured using a fluorescence microscope (a Zeiss LSM 880 model, manufactured in Oberkochen, Germany).

Statistical analysis

The software application known as Prism was employed for statistical analysis, whereas the independent Student’s t-test was applied to assess the data from the two distinct groups. When dealing with comparisons across three or more distinct groups, a single-factor analysis of variance (ANOVA) was conducted, succeeded by a Least Significant Difference (LSD) test. A P value less than 0.05 was deemed to indicate statistical significance. Replications of the tests for each experimental cohort were conducted thrice. The data from triplicate experiments (technical replicates) were represented as average values ± standard error of the mean (SEM).

Results

Expression of RHOJ in melanoma cells

To explore RHOJ expression in melanoma and melanocyte cells, we examined the GEO dataset GSE122907, which encompasses data from both melanoma and melanocyte samples from identical patients. Analysis using a volcano plot demonstrated that RHOJ levels were markedly elevated in melanoma cells relative to normal melanocytes (Figure 1A). Additional investigation through the GEPIA database assessed RHOJ expression in melanoma tissues. As depicted in Figure 1B, notable disparities were found between the control group and the model group, with upregulated elements shown in red and downregulated ones in gray. Moreover, A375 cells displayed the highest RHOJ expression (P<0.001), followed by A2058 and HS294T cells, while PIG1 cells exhibited the lowest expression levels (Figure 1C,1D). These results further validate the differential expression of RHOJ in melanoma cells, offering valuable molecular insights into its potential role in melanoma progression.

Figure 1 Expression of RHOJ in melanoma cells. (A) Expression analysis of melanoma and melanocytes from the same patient using the GEO dataset GSE122907. (B) Analysis of RHOJ expression levels in melanoma tissues using the GEPIA database. Red: tumor tissues; grey: normal tissues. (C) RT-qPCR detection of RHOJ expression levels in melanoma cells. (D) Western blot analysis of RHOJ expression levels in melanoma cells. Data are presented as mean ± SD. *, P<0.05; **, P<0.01; ***, P<0.001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GEO, Gene Expression Omnibus; GEPIA, Gene Expression Profiling Interactive Analysis; RT-qPCR, real-time quantitative reverse transcription polymerase chain reaction; SD, standard deviation; SKCM, skin cutaneous melanoma; TPM, transcripts per million.

Effect of knockdown RHOJ on proliferation and apoptosis of melanoma cells A375

Following transfection with si-RHOJ, RHOJ expression significantly decreased (P<0.001), confirming successful gene knockdown and setting the stage for further investigation into RHOJ’s involvement in cellular functions and signaling pathways (Figure 2A,2B). Cell viability was assessed using the CCK-8 assay, which showed a reduction in A375 cell viability 48 hours post-si-RHOJ transfection (P<0.001, Figure 2C). Cell proliferation was evaluated through the EdU assay, revealing diminished proliferation in cells transfected with si-RHOJ for 48 hours (P<0.05, Figure 2D). Flow cytometry analysis of cell apoptosis demonstrated increased apoptosis in cells with si-RHOJ transfection for 48 hours (P<0.001, Figure 2E). Additionally, Western blot analysis indicated upregulation of Bax and downregulation of Bcl-2 in A375 cells with si-RHOJ transfection (P<0.001, Figure 2F). These changes in apoptosis-related proteins underscore RHOJ’s potential role in modulating apoptotic pathways in melanoma cells, highlighting its importance as a prospective therapeutic target for melanoma treatment.

Figure 2 Effects of RHOJ knockdown on proliferation and apoptosis of A375 cells. (A) Efficiency of RHOJ knockdown assessed by RT-qPCR. (B) Efficiency of RHOJ knockdown assessed by Western blot. (C) Impact of RHOJ knockdown on the viability of melanoma cells. (D) Effect of RHOJ knockdown on proliferation of A375 cells assessed by EdU assay (200×). (E) Effect of RHOJ knockdown on apoptosis of melanoma cells. (F) Impact of RHOJ knockdown on apoptosis-related proteins in melanoma cells. Data are presented as mean ± SD. *, P<0.05; ***, P<0.001. EdU, 5-ethynyl-2'-deoxyuridine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; OD, optical density; RT-qPCR, real-time quantitative reverse transcription polymerase chain reaction; SD, standard deviation.

Impact of RHOJ knockdown on adhesion of melanoma cells A375

This investigation utilized a cell adhesion assay kit to assess how RHOJ gene knockdown affects cell adhesion. There was a notable reduction in cell adhesion in A375 cells transfected with si-RHOJ (P<0.001, Figure 3A). To evaluate the expression of adhesion proteins, immunofluorescence was performed, focusing on cell-cell adhesion protein E-cadherin and cell-matrix adhesion protein vinculin. Post-transfection with si-RHOJ, we observed an increase in E-cadherin levels and a decrease in vinculin in A375 cells (Figure 3B). Additionally, Western blot analysis was conducted to measure the levels of E-cadherin and vinculin. Results showed that E-cadherin was upregulated and vinculin was downregulated in A375 cells following si-RHOJ transfection (P<0.001, Figure 3C). These findings indicate that RHOJ knockdown directly affects cell-matrix adhesion.

Figure 3 Impact of RHOJ knockdown on adhesion capability of A375 cells. (A) Cell adhesion assay using a cell adhesion detection kit. (B) Immunofluorescence detection of cell-cell adhesion (E-cadherin) and cell-matrix adhesion protein (vinculin) expression (200×). (C) Western blot analysis of cell-cell adhesion (E-cadherin) and cell-matrix adhesion protein (vinculin) expression. Data are presented as mean ± SD. ***, P<0.001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SD, standard deviation.

Effect of knocking down RHOJ on the Rap1 signaling pathway

PPI network showed that 10 genes were closely related to RHOJ by STRING database (https://cn.string-db.org/) (Figure 4A). These genes also lay a theoretical foundation for future studies exploring the potential implications of RHOJ in disease development. After Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, the above 10 genes mainly enriched in the Rap1 signaling pathway (Figure 4B). RAP1, RAP1GAP, and RasGRP3 are key proteins in the Rap1 signaling pathway. The expression of the above proteins was detected by Western blot. The protein levels of RAP1, RAP1GAP, and RasGRP3 were decreased in A375 cells transfected with si-RHOJ (P<0.001), however, they reversed by the Activator of the Rap1 signaling pathway (P<0.001, Figure 4C). These results suggest a potential role for RHOJ in regulating the Rap1 signaling pathway, providing important insights for further exploration of its molecular mechanisms and implications in disease.

Figure 4 Influence of RHOJ knockdown on the Rap1 signaling pathway. (A) Analysis of RHOJ-related genes using the String database. (B) KEGG pathway enrichment analysis of RHOJ and its related genes using the String database. (C) Western blot analysis of the effects of the Rap1 signaling pathway-related proteins RAP1, RAP1GAP, and RasGRP3. Data are presented as mean ± SD. ***, P<0.001 vs. Control group; ^###, P<0.001 vs. si-RHOJ group. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; KEGG, Kyoto Encyclopedia of Genes and Genomes; SD, standard deviation.

Impact of RHOJ knockdown via Rap1 signaling pathway on proliferation and apoptosis of melanoma cells A375

It was observed that A375 melanoma cells exhibited diminished cellular activity in the si-RHOJ group, while a significant increase in activity was seen in the si-RHOJ + Activator group (P<0.001, Figure 5A). The data indicated a substantial increase in the proportion of EdU-positive cells in the si-RHOJ + Activator group compared to the si-RHOJ group (P<0.05, Figure 5B). Furthermore, the apoptosis rate was markedly lower in the si-RHOJ + Activator group compared to the si-RHOJ group for A375 melanoma cells (P<0.001, Figure 5C). Additionally, expression levels of Bax were significantly reduced, whereas Bcl2 expression was markedly increased in the si-RHOJ + Activator group compared to the si-RHOJ group (P<0.001, Figure 5D). These findings imply that RHOJ knockdown may affect the apoptosis process in A375 cells by altering Bax and Bcl2 protein levels, with activator treatment potentially mitigating these effects.

Figure 5 Effects of RHOJ knockdown via the Rap1 signaling pathway on proliferation and apoptosis of A375 cells. (A) CCK-8 assay to assess the impact of RHOJ knockdown on the viability of A375 cells. (B) EdU assay to evaluate the effect of RHOJ knockdown on the proliferation of A375 cells (200×). (C) Flow cytometry to detect the influence of RHOJ knockdown on apoptosis of A375 cells. (D) Western blot analysis to examine the effect of RHOJ knockdown on apoptosis-related proteins in A375 cells. Data are presented as mean ± SD. *, P<0.05; ***, P<0.001. CCK-8, Cell Counting Kit-8; EdU, 5-ethynyl-2'-deoxyuridine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SD, standard deviation.

Effect of knockdown RHOJ on the adhesion of melanoma cells A375 through Rap1 signaling pathway

Experimental data revealed that the cell adhesion rate was notably higher in the si-RHOJ + Activator group compared to the si-RHOJ group (P<0.01, Figure 6A). In the si-RHOJ + Activator group, there was a significant reduction in E-cadherin expression, while vinculin expression was markedly elevated (Figure 6B) in the Immunofluorescence detection. Additionally, E-cadherin expression significantly dropped and vinculin expression substantially increased in the si-RHOJ + Activator group in the Western blot analysis (P<0.001, Figure 6C). These observations suggest that knocking down RHOJ might impact cell-cell and cell-matrix adhesion properties by altering the levels of E-cadherin and vinculin.

Figure 6 Impact of RHOJ knockdown via the Rap1 signaling pathway on adhesion capability of A375 cells. (A) Performing cell adhesion assay using a cell adhesion detection kit. (B) Immunofluorescence detection of cell-cell adhesion (E-cadherin) and cell-matrix adhesion protein (vinculin) expression (200×). (C) Western blot analysis of cell-cell adhesion (E-cadherin) and cell-matrix adhesion protein (vinculin) expression. Data are presented as mean ± SD. **, P<0.01; ***, P<0.001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SD, standard deviation.

Discussion

Our initial immunofluorescence experiments demonstrated that direct knockdown of RHOJ did not result in changes in the expression levels of E-cadherin and vinculin compared to control conditions. However, when we introduced an activator specific to RHOJ, we observed a notable reduction in E-cadherin expression, along with a significant increase in vinculin expression (19). Recent studies (8,20,21) have shown significant overexpression of RHOJ in glioblastoma, in which it promotes angiogenesis and tumor invasion. Additionally, upregulation of RHOJ enhances invasion and metastasis in epithelial-to-mesenchymal transition (EMT)-subtype gastric cancer through the IL-6/STAT3 signaling pathway (22). Members of the Rho GTPase family play a crucial role in orchestrating a range of cellular functions in human melanoma, including tumor initiation, the dissemination of metastatic cells, and resistance to chemotherapeutic agents. RHOJ influences the reorganization of the actin cytoskeleton by activating the phosphorylation of LIMK, cofilin, and p41-ARC (a component of the ARP2/3 complex) through a mechanism dependent on PAK1 activity, as observed in both cellular assays and orthotopic tumor models. However, further research is needed to fully understand the role of RHOJ in melanoma. Experimental studies using in vitro models and tumor xenografts have provided mechanistic insights into RHOJ’s function. Specifically, RHOJ-mediated phosphorylation events are dependent on PAK1, a kinase known for its role in cell motility and cancer progression. Our study confirms that RHOJ is significantly upregulated in melanoma cells compared to normal melanocytes, a finding consistent with other research (23), which is also consistent with other studies (24,25) that reported increased RHOJ expression in various cancers, including melanoma. Elevated RHOJ levels in melanoma suggest its potential involvement in tumor progression, further supporting previous research that identified RHOJ as a marker for cancerous tissues.

The knockdown of RHOJ resulted in decreased cell viability and proliferation, along with increased apoptosis. These results are consistent with findings from a previous study (23), which observed similar effects in breast cancer cells following RHOJ silencing. The increase in Bax and the decrease in Bcl-2 levels, as noted in our study, highlight RHOJ’s role in regulating apoptosis. This modulation of apoptotic pathways is crucial for understanding how RHOJ affects cancer cell survival and supports the idea that RHOJ could be a viable therapeutic target.

Activation of RAP1 signaling is associated with aggressive phenotypes in malignant tumors. In prostate cancer, glioblastoma, non-small cell lung cancer, melanoma, breast cancer, and pancreatic cancer, RAP1 has exhibited oncogenic activity by promoting tumor cell invasion and migration. Additionally, RAP1 has been implicated in the transcriptional regulation of metabolic pathways (26,27). Rap1 activity is responsible for the activation of extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) signaling in ovarian cancer. Rap1A activates the p38 pathway, enhancing the expression of several EMT markers, thereby promoting tumorigenesis and metastasis. EMT is recognized as a prerequisite for tumor invasion and ultimate metastasis. Among the key regulators, transforming growth factor-beta (TGF-β) exhibits extensive signaling crosstalk with various Rho GTPases. In particular, Rho and Rac1 exhibit synergistic effects during EMT in both normal and tumor epithelial cells (28). Kamran Hosseini et al. discovered, in breast epithelial carcinoma cells (MCF-7), that EMT leads to characteristic changes in the cortical association of Rho-GTPases, including Rac1, RhoA, and RhoC, as well as downstream actin regulators such as cofilin, mDia1, and Arp2/3. They proposed that the interaction between Rac1 and RhoC serves as one of the signaling pathways underlying the cortical mechanical changes induced by EMT (29). Satoh et al. found that inhibiting RhoQ expression promotes TGF-β-mediated EMT and invasion in lung adenocarcinoma (LUAD) cell lines, and that low RhoQ levels are associated with poorer overall survival in LUAD patients (30). Additionally, Rho-associated kinase-mediated EMT facilitates metastasis in laryngeal squamous cell carcinoma (31). Moreover, Rho proteins can also participate in EMT development and tumorigenesis through their upstream regulators, Rho GTPase-activating proteins (Rho-GAPs) (32). These studies suggest that RHOJ may also be involved in the regulation of melanoma cell adhesion through mechanisms beyond the Rap1 signaling pathway. Further research is warranted to elucidate the relevant signaling pathways and upstream-downstream mechanisms to identify the key players involved.

To validate and further explore these findings, we employed Western blotting, which confirmed the immunofluorescence results. Specifically, under conditions where RHOJ was activated, there was a significant decrease in E-cadherin expression and a marked increase in vinculin expression. These results highlight the dynamic and context-dependent nature of RHOJ’s regulatory functions in modulating adhesion protein expression, which are critical for maintaining cellular integrity, cytoskeletal organization, and intercellular interactions (33,34). The implications of our findings extend beyond basic cell biology to the realm of cancer biology. Dysregulation of cell adhesion processes is a hallmark of cancer progression and metastasis. The observed effects of RHOJ on E-cadherin and vinculin expression provide valuable insights into potential therapeutic strategies aimed at targeting adhesion-related pathways in cancer cells (35,36). For instance, therapies that modulate RHOJ activity or its downstream signaling pathways could potentially influence tumor cell adhesion properties and invasive behavior, offering novel avenues for therapeutic intervention in cancer treatment (10,25). RHOJ has been shown to regulate melanoma cell migration and invasion by influencing cytoskeletal dynamics and cell adhesion molecules. This pathway contributes to tumor progression by affecting cellular motility and interactions. Meanwhile, the Rap1 signaling pathway plays a crucial role in maintaining cell-cell junctions and modulating cell adhesion, which impacts tumor cell dissemination and metastasis. Advances in these areas offer promising avenues for developing targeted therapies to address melanoma progression and metastasis. However, the study does not include in vivo validation of the findings. Animal models or patient-derived xenografts could provide more comprehensive insights into the role of RHOJ in melanoma progression and treatment responses.

Moreover, this study enhances our understanding of RHOJ as a critical regulator of cell adhesion dynamics, highlighting its potential as a therapeutic target in cancer and other diseases characterized by dysregulated cell adhesion. By elucidating the complex interplay between RHOJ and key adhesion molecules like E-cadherin and vinculin, we pave the way for future research aimed at developing targeted therapies and improving clinical outcomes in cancer treatment and beyond.

Conclusions

This study reveals that RHOJ regulates cell adhesion by modulating key proteins E-cadherin and vinculin. While RHOJ knockdown initially showed no significant effect, activation of RHOJ led to decreased E-cadherin and increased vinculin levels, indicating its role in both cell-cell and cell-matrix adhesion. These findings, combined with RHOJ’s involvement in EMT-related chemotherapy resistance, suggest its potential as a therapeutic target in cancer treatment and tumor vasculature targeting.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2024-2692-b/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2024-2692-b/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2024-2692-b/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2024-2692-b/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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