CCL5 promotes colon cancer progression via PI3K/AKT signaling and is negatively regulated by STAT1

Introduction

According to research, colon cancer (CC) is one of the top three most prevalent cancers in the US in terms of incidence and death (1). CC is one of the most common malignancies in China, the most populous country in the world (2). It makes up around 10% of all cancer diagnoses in the nation, with over 370,000 cases reported each year (3). Despite advances in medicine, the prevalence of CC has not decreased as much as other cancers, and it still accounts for around 10% of all cancer cases (4). In the meantime, immunotherapy has made significant strides in the treatment of cancer, including CC patients (5). According to clinical research, the immune response in cancer patients begins when immune cells are drawn into the tumor microenvironment (TME) to start immune-mediated tumor cell killing (6). Cancer cells undergo reprogramming and release chemokines that draw T lymphocytes, monocytes, myeloid cells, fibroblasts, and mesenchymal stromal cells from bone marrow or adipose tissue in order to promote tumor growth, proliferation, and metastasis (7). One of these cytokines is CCL5, a member of the CC-chemokine subfamily and also referred to as Regulated upon Activation, Normal T-cell Expressed and Secreted, or RANTES (8). T lymphocytes and monocytes, in particular, are immune cells that express CCL5. This multifunctional chemokine binds to several receptors, such as CCR1, CCR3, CCR4, and CCR5; the maximum binding affinity is shown by CCR5, a seven-transmembrane G protein-coupled receptor (GPCR) (9). According to current studies, CCL5 has two roles in cancer. On the one hand, it promotes anti-tumor immunity by attracting anti-tumor T cells and dendritic cells into the TME, which improves immunotherapeutic responses across a variety of tumor types (10,11). However, CCL5 can potentially affect tumor growth by means of both paracrine and autocrine processes. By recruiting inflammatory cells by paracrine activity, it indirectly shapes the TME while directly controlling the migration, survival, and proliferation of cancer cells through autocrine signaling. For instance, it helps create a cancer microenvironment that is conducive to survival (12).

The dual function of CCL5 has been further clarified by CC research. In comparison to controls, studies employing mice lacking CCL5 showed a much higher infiltration of CD8⁺ T cells in initial CC tumors. Programmed cell death protein 1 (PD-1) and its ligand PD-L1 are key molecules in the immune checkpoint pathway. PD-L1 is expressed on the surface of tumor cells, and its binding to PD-1 on the surface of T cells can inhibit anti-tumor immune responses and promote immune evasion. Increased PD-1/PD-L1 expression, decreased sensitivity to anti-PD-1 treatment, and metabolic disturbance of tumor-associated macrophages in the TME have all been linked to CCL5 deficiency, which ultimately promotes CD8⁺ T cell migration (13). On the other hand, it was discovered that CCL5 generated from CC tumor buds attracts fibroblasts by binding to CCR5. In order to encourage the advancement of CC, this interaction may activate p-Akt-p-mTOR signaling by upregulating fibroblast expression of solute carrier family 25 member 24 (SLC25A24). Furthermore, by promoting VEGF expression, fibroblast differentiation into vascular endothelial cells, and collagen synthesis to modify the TME, CCL5 may aid tumor angiogenesis (14). Other research shows that CCL5 has anti-tumor effects in CC, which contradicts our findings. Immune cells that are essential for anti-tumor immunity, such as CD8⁺ T cells and type 1 dendritic cells (cDC1s), are recruited by CCL5. When paired with activation treatments, their tumor invasion may improve the response to immunotherapy (11). Higher expression is linked to increased cDC1 presence and better patient survival, indicating that the CCL5/CCR5 axis is crucial for cDC1 infiltration (15).

In conclusion, CCL5 plays roles in CC that depend on context. There are contradictory findings on the relationship between CCL5 expression and clinical outcomes, despite some research emphasizing its chemokine role in immune cell recruitment. In cetuximab-combined treatment, low CCL5 expression has been linked to improved survival (16), and mechanistic research demonstrates direct tumor growth suppression (17). However, through the recruitment of immunosuppressive cells [myeloid-derived suppressor cells (MDSCs), macrophages, and regulatory T cells (Tregs)], increased CCL5 is associated with a poor prognosis (18,19). These differences highlight the necessity of more research on the direct impact of CCL5 on CC cells. Our research fills this gap by using both in vitro and in vivo trials to investigate the connection between CCL5 expression and CC cell behavior. PI3K/AKT pathway activation mediated by CCL5 was discovered using transcriptome sequencing. Dual luciferase reporter experiments validated our identification of STAT1 as a putative upstream regulator of CCL5 by protein interaction network analysis and public database prediction. Subsequent research revealed that STAT1 suppresses the production of CCL5. We present this article in accordance with the MDAR and ARRIVE reporting checklists (available at https://jgo.amegroups.com/article/view/10.21037/jgo-2025-aw-917/rc).

Methods

This combined retrospective clinical analysis with experimental validation. Human tissues (n=40) were collected via convenience sampling. Immunohistochemistry and public databases [The Cancer Genome Atlas (TCGA)/Genotype-Tissue Expression (GTEx)] were used for clinical correlation. Experimental tools included cell functional assays [quantitative reverse transcription polymerase chain reaction (qRT-PCR), Cell Counting Kit-8 (CCK-8), Transwell, scratch tests], transcriptome sequencing, dual-luciferase reporter assays, and nude mouse xenograft models. Detailed protocols are described below.

Immunohistochemistry

The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethical Committee of The First Affiliated Hospital of Kunming Medical University [IRB: (2020) Ethics L No. 159; approval date: March 8, 2024]. Verbal informed consent was obtained from all participants or their family members via telephone prior to the use of their archived tissue specimens for research. Additionally, we were instructed to handwrite down the contents of the phone conversation. The tissue samples were wax block tissues from Department of Pathology, The First Affiliated Hospital of Kunming Medical University, as well as samples from patients who had been given a CC diagnosis based on their admission medical records and postoperative pathology. Standardized treatment for CC was given to each of these individuals. For a retrospective investigation, we gathered post-operative tissues. Between January 2024 and March 2024, we gathered 40 tissue wax block cases without any prior radiation.

Following standard dewaxing, antigen retrieval, and blocking procedures, sections were incubated with primary antibody overnight at 4 ℃ and HRP-labeled secondary antibody for 50 min at room temperature. DAB visualization and hematoxylin counterstaining were performed.

CCL5 expression (cytoplasmic/membranous) was evaluated independently by two pathologists using the Fromowitz criteria (20). The percentage of positive cell counts is denoted by A: A ≤1%, 0; 1%< A ≤10%, 1; 10%< A ≤50%, 2; 51%< A ≤75%, 3; 75%< A ≤100%, 4; B stands for the staining intensity percentage: tan, 2; yellow-brown-tan, 3; pale yellow, 1; and no staining, 0. A×B ≥2, indicating a positive CCL5. Score: negative, 0–2; weakly positive (+), 2–3; moderately positive (++), 3–4; very positive (++++, ++++), 5–12 (21). To find the staining index, multiply these two values; a staining index of 8 indicates high expression; a value of less than 8 indicates low expression.

Analysis of CCL5 in public databases and patient prognoses for CC

TCGA (https://cancergenome.nih.gov/) was searched to find the transcriptome expression count data of CCL5 in human CC tissue and normal tissue samples. The R program was used to examine the levels of CCL5 mRNA expression in primary tumor tissues and normal tissues.

The Kaplan-Meier Plotter database (https://kmplot.com) was used to examine the correlation between the overall survival time of CC patients and high and low CCL5 gene expression.

Cell lines and culture

Prosperity Life Science and Technology Co., Ltd. (Wuhan, China) supplied the human CC cell lines SW480, RKO, CW-2, HT-29, and mouse CC cells CT-26. The cells’ basal media were DMEM (Pricella, Wuhan, China) for SW480, RKO, CW-2, and CT-26, and McCoy’s 5A medium (Gibco, Carlsbad, CA, USA) for HT-29 cells. Following the addition of 10% FBS and 1% P/S to the basal medium, the cells were cultivated at 37 ℃ in an incubator with 5% CO₂.

Lentivirus infection

We infected the target cells with lentiviral particles containing GFP-Puro CCL5 short hairpin RNA (shRNA) and overexpression of the CCL5 gene in order to produce stable CCL5 overexpression and knockdown cell lines (HanBio, Shanghai, China). In short, lentivirus-infected 1.5×10⁵ tumor cells were plated on 6-well plates with 1 mL of media [10 multiplicity of infection (MOI)], and the medium was replenished after 4 hours. After being incubated throughout the entire night at 37 ℃ with 5% CO₂, the medium was changed. Appendix 1 and Table S1 display the sequencing of the target genes.

qRT-PCR

Following the manufacturer’s recommendations, total cellular RNA was extracted from cells using Invitrogen’s TRIzol reagent. The All-In-One 5X RT Master Mix (abm, Vancouver, Canada) was then used to reverse transcribe the resulting cDNA. Then, using a Roche LightCycler^® 480 System (Roche, Indianapolis, USA), total cellular RNA was extracted from cells for qPCR using the Prime Script^TM RT reagent Kit (Chengdong District, Osaka City, Japan). The following amplification conditions were established: 30 s at 95 ℃, 1 cycle of 95 ℃ for 5 s, and 30 s at 60 ℃ for 40 cycles. After normalizing the mRNA levels to those of GAPDH, the 2^−ΔΔCt technique was used to determine each mRNA’s relative expression. In Table 1, the primer sequences are displayed.

Colony formation assay

The cells of the stable transplants in the logarithmic growth phase were digested by trypsin, then resuspended in full media and counted. Following the inoculation of 1,500 cells per group in a six-well plate, 2 mL of complete media was added, the cells were thoroughly mixed, and the six-well plate was placed in an incubator set at 37 ℃ with 5% CO₂ to continue the culture. Keep the culture going for two to three weeks. After the cloning process is finished, discard the old medium, wash with PBS once, fix each well with 1 mL of 4% paraformaldehyde for 30 minutes, aspirate the fixative, wash with PBS once, and let it air dry. Then, add 1 mL of crystal violet staining solution to each well, stain for 10 minutes, and wash with PBS twice. To count the clone sites with cell numbers more than 50 in the clones, the six-well plate was put under an inverted white light microscope. The clones were then photographed for storage.

Viability of cells CCK-8 test

CCK-8 was used to measure changes in cell viability. Specifically, 96-well plates were inoculated with 3,000 cells/well of the stable transfected strain CC cells (24 hours after transfection) in 100 µL of complete media, and the plates were then incubated at 37 ℃. After adding 10 µL of CCK-8 reagent (Beyotime, Shanghai, China) to each well at the conclusion of each experiment, the cells were incubated for an additional two hours at 37 ℃. A Multiskan microtiter plate device (Thermo Fisher Scientific, Waltham, MA, USA) [optical density (OD)450] was then used to measure the optical density readings.

Cell chamber migration and invasion assay

To evaluate cell migration and invasion capacity, Transwell experiments were conducted in a 24-well plate using Transwell chambers (Corning Incorporated, Corning, NY, USA) with an 8 µm pore size. 600 µL of complete medium containing 15% FBS was inoculated in the lower chamber of the cell migration test, whereas 200 µL of basal medium without FBS was used to inoculate 1×10⁴ cells in the upper chamber. For 48 hours, the cells were incubated at 37 ℃. A cotton swab was used to remove the remaining cells from the top of the filter. Cells moving to the opposite side of the filter, meanwhile, were fixed for 20 minutes at room temperature using 4% paraformaldehyde (Biosharp, Hefei, China) and then stained for 20 minutes using 0.1% crystal violet (Servicebio, Wuhan, China).

50 µL of Matrigel (BD Bioscience, San Jose, CA, USA) was used to pre-coat the filters for the invasion studies. These filters were then diluted with ice-cold serum-free media at a ratio of 1:8, incubated for three hours in Transwell chambers, then hydrated for thirty minutes at 37 ℃. The remaining experimental procedures were identical to those used in the Transwell migration studies of tumor cells.

Wound‑healing assay

In a 6-well plate, 2 mL of media containing 10% fetal bovine serum was used to inoculate the gene-transfected cells, which were then grown for 24 hours. A 200 µL sterile pipette tip was used to scrape the cells vertically once the cell density exceeded 95%. After carefully washing away the floating cells with phosphate-buffered saline, the cells were grown for 48 hours in 2 mL of serum-free media. A light microscope (Olympus, Tokyo, Japan) with a magnification of ×40 was used to acquire pictures of cells at 0 and 48 hours in three different random fields of view for every experimental setup.

Transcriptome sequencing

RNA sequencing was performed on RKO which was confirmed to have successfully expressed the CCL5 gene as previously mentioned. TRIzol reagent was used to separate the cells’ total cellular RNA, and the resulting RNA concentration and quality were found to be satisfactory.

Library preparation for Transcriptome sequencing

To purify mRNA, we utilized magnetic beads with poly-T oligo attachments. Lysis was carried out at high temperatures using divalent cations in first-strand synthesis reaction buffer (5×). M-MuLV reverse transcriptase and random hexamer primers were used to create first-strand cDNA, which was subsequently broken down by RNaseH. DNA polymerase I and dNTP were used to create the second strand of cDNA. The exonuclease/polymerase activity transformed the remaining overhangs into blunt ends. An adaptor with a hairpin loop structure was ligated and made ready for hybridization following the adenylation of the DNA fragment’s 3' end. The AMPure XP system (Beckman Coulter, Beverly, USA) was used to purify library fragments in order to preferentially pick cDNA fragments that were between 370 and 420 bp in length. The library was then obtained by purifying the PCR products using AMPure XP beads after PCR amplification. Following the completion of library construction, the library was first measured using a Qubit 2.0 fluorometer. It was then diluted to 1.5 ng/µL, and an Agilent 2100 Bioanalyzer was used to determine the size of the library insert. Once the insert size was as anticipated, the library’s effective concentration (>2 nM) was precisely measured using qRT-PCR to guarantee its quality.

Transcriptome sequencing

Following the qualification of the clustering and sequencing libraries, Illumina NovaSeq 6000 was used for sequencing.

Data analysis

Using the DESeq2 R software (3.20.0), differential expression analysis was carried out on cells that were successfully overexpressing CCL5 as well as control cells (3 biological replicates per condition). DESeq2 offers statistical procedures for detecting differential expression in numerical gene expression data by using a model based on the negative binomial distribution. The threshold for substantial differential expression is |log₂fold change (FC)| >1 and P<0.05.

The clusterProfiler R package (3.14.3) was used to implement the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analysis of differentially expressed genes (DEGs). For differential genes, corrected P values below 0.05 were regarded as significantly enriched.

Tumor mouse model establishment

A protocol was prepared before the study without registration. All animal experiments were performed under a project license (No. kmmu20240669) granted by the Animal Ethics Committee of Kunming Medical University, in compliance with institutional guidelines for the care and use of animals. Twenty female nude mice (15±1.2 g, 4 weeks) were purchased from Yunnan Bethesda Biological Company and housed under specific pathogen‑free conditions (temperature 26–28 ℃, humidity 40–60%, 10 h light/14 h dark cycle) with free access to food and water. After one week of acclimation, the mice were randomly divided into four groups (n=5). Stable CT-26 (overexpression) and HT‑29 (knockdown) cell lines in logarithmic growth phase were suspended in PBS (1×10⁶–5×10⁶ cells/100 µL) and subcutaneously injected into the right axilla of each mouse. Tumor length and width were measured twice weekly by a blinded investigator, and volume was calculated as V = 0.5 × length × width². Humane endpoints were strictly observed, including tumor size exceeding 10% of body weight or reaching a maximum diameter of 15 mm, along with signs of severe distress (immobility, unresponsiveness, cyanosis, or dyspnea). Mice were euthanized by intraperitoneal injection of pentobarbital sodium (1 mL/kg) after 3 weeks (CT-26) or 4 weeks (HT-29), and final tumor volumes were recorded.

Using transcriptomics data from this experiment and public databases to predict CCL5 upstream regulatory factors

In this study, we used several databases to predict possible transcription factors of the CCL5 gene in CC patients. In order to determine the possible upstream transcription factors of the final CCL5, Pearson correlation analysis was carried out using the data profile of colon adenocarcinoma (COAD) patients from the TCGA database as the data source. The correlation coefficient was set at 0.3, the |log₂FC| threshold in KnockTF was set at 0.5, and the results were displayed as Venn diagrams. Among these databases are: hTFtarget (http://bioinfo.life.hust.edu.cn/hTFtarget#!/), CHEA (https://maayanlab.cloud/chea3/), ENCODE (https://maayanlab.cloud/Harmonizome/dataset/ENCODE+Transcription+Factor+Targets) and KnockTF (https://bio.liclab.net/KnockTF/index.php).

Assay for dual luciferase reporters

Assay for luciferase activity

CC cells should be divided into 24-well plates with a cell density of 70% to 80% in order to be ready for transfection. Solution A involves thoroughly mixing 100 µL DMEM with 1.2 µg of the target plasmid (PRL: 0.2 µg, promoter: 0.4 µg, transcription factor: 0.6 µg) and letting it sit at room temperature. Solution B involves mixing 100 µL DMEM with 2 µL of transfection reagent (HanBio product with a concentration of 0.8 mg/mL). After thoroughly mixing solutions A and B, they were allowed to sit at room temperature for 20 minutes. After discarding the medium and substituting it with new medium, the transfection mixture was added and thoroughly mixed. After that, the samples were kept in an incubator with 5% CO₂ at 37 ℃. After 48 hours of incubation, gather the samples for analysis.

Assay for firefly fluoresceinase

After the 24-well plate was taken out of the incubator, it was left at room temperature until the cells had reached room temperature. To completely lyse the cells, add 500 µL of the Passive Lysis Buffer reagent to each well, blow up the cells with a pipette gun, and then let it sit at room temperature for 10 minutes. After centrifuging the cells for 10 minutes at 12,000 rpm and 4 ℃, transfer 40 µL to 96 wells. To record the Firefly luciferase value, add 50 µL of LAR II to each well and place it in the enzyme marker. After that, the samples were kept in an incubator with 5% CO₂ at 37 ℃. After 48 hours of incubation, gather the samples for analysis. To record the Renillaluc luciferase value, add 50 µL of Stop & Glo^® Reagents to each well and place it in the enzyme marker. Firefly luciferase value/Renillaluc luciferase value is the experiment's final numerical result.

Statistical analysis

IBM SPSS 25 scientific research software was used to statistically analyze all of the data. The scientific research program Graphad Prism 9 was used to draw the data. This study included forty CC patients who were categorized into high and low expression groups based on immunohistochemical scoring results. The group with high expression CCL5 was defined as having a score greater than 8, and the group with low expression CCL5 was defined as having a score less than 8. Fisher’s exact probability method was then used to evaluate the clinical data of the two groups of patients with high and low expression of CCL5 proteins. The two independent samples t-test was used to statistically examine comparisons between the mouse groups. The experimental procedure is homogeneous, we employ randomized groups, and the data results are typically regularly distributed. A two-sided test was used to examine all of the research, and a difference was deemed statistically significant when P<0.05.

Results

Variations of CCL5 expression in CC patients and prognosis

This study discovered, using the public database, that CCL5 was statistically considerably lower in CC cancer tissues than in TCGA database paracancer tissues. This study also compared the expression of the CCL5 gene in normal intestinal tissues in the GTEx database with that in cancer tissues in the TCGA database using the GEPIA2 database because the TCGA database compares cancer and paracancerous tissues. The study’s comparison of normal intestinal tissues revealed that CC cancer tissues had much higher levels of CCL5 expression, with a statistically significant difference. According to the analysis, the order of CCL5 expression in CC tissues is as follows: normal intestinal tissues < cancer tissues < paracancer tissues. The aforesaid results were corroborated by the immunohistochemistry scoring results.

According to the results obtained from the public database, CC patients with high CCL5 gene expression had a statistically significant longer overall survival time than CC patients with low CCL5 gene expression. Figure 1 displays the findings.

Figure 1 CCL5 expression is upregulated in colon cancer and associated with patient prognosis. (A) Immunohistochemical findings of CCL5 in distal, paracancerous, and CC tissues. (B) Analyzing immunohistochemistry results quantitatively. (C) CCL5 expression in colon cancer, paracancerous, and normal intestinal tissues was determined by transcriptome sequencing. (D) CCL5 expression levels and the prognosis of patients with CC. *, P<0.05; ****, P<0.0001. CC, colon cancer; CI, confidence interval; COAD, colon adenocarcinoma; HR, hazard ratio; IHC, immunohistochemical; TPM, transcript per million.

Analysis of the connection between CCL5 expression and CC patients’ clinicopathological information

The Fisher’s exact test was utilized in this study to examine the differences between the two groups because there were 40 samples in total. The comparison showed that the degree of tumor differentiation varied between those with high and low CCL5 expression, but that age, gender, maximal tumor margin diameter, clinical stage, T stage, and lymph node metastasis did not significantly differ. Table 2 displays the findings.

CCL5 expression’s impact on CC cells’ biological activity in vitro

In vitro, CC cell invasion, migration, and proliferation are enhanced by CCL5 overexpression

To create stable CCL5 overexpression lines, we infected RKO and SW480 cells with lentivirus containing the overexpressed human CCL5 gene and empty vector group (EG). Figure 2A shows the plasmid vector we constructed for the overexpression of lentivirus. Levated CCL5 expression was confirmed by qRT-PCR results (Figure 2B). Furthermore, we conducted the Transwell tumor cell migration and invasion experiment, scratch assay, plate clone formation assay, and cell viability CCK-8 assay. According to the plate clone formation assay and CCK-8, our data demonstrated that up-regulation of CCL5 expression enhanced the proliferative activity of RKO and SW480 cells (P<0.05, Figure 2C,2D). The migratory and invasive activity of RKO and SW480 cells was enhanced by up-regulation of CCL5 expression (P<0.05, Figure 2E-2G).

Figure 2 CCL5 overexpression promotes colon cancer cell proliferation, migration, and invasion in vitro. (A) Schematic representation of the lentiviral vector for human CCL5 overexpression. (B) qRT-PCR validation of CCL5 mRNA expression in RKO and SW480 cells after lentiviral infection. (C) Colony formation assay and quantitative analysis of colony numbers (crystal violet staining). (D) CCK-8 cell viability assay showing proliferative capacity of RKO and SW480 cells over 4 days. (E) Representative images of scratch wound healing assay at 0 and 48 hours (crystal violet staining, ×40) and quantitative analysis of wound closure rate. (F) Transwell migration assay (crystal violet staining, ×40) and quantitative analysis of migrated cells. (G) Transwell invasion assay (Matrigel-coated; crystal violet staining, ×40) and quantitative analysis of invaded cells. Data are presented as mean ± standard deviation. *, P<0.05; ***, P<0.001; ****, P<0.0001. CCK-8, Cell Counting Kit-8; NC, negative control; OD, optical density; OE, overexpression; qRT-PCR, quantitative reverse transcription polymerase chain reaction.

In vitro, CC cell invasion, migration, and proliferation are inhibited by CCL5 knockdown

To create stable CCL5 knockdown strains, we infected CW-2 and HT-29 cells with EG and lentivirus that carried the CCL5 gene knockdown. After evaluating alterations in tumor cell invasion, migration, and proliferation, we discovered that CC cells in vitro were negatively impacted by downregulating CCL5 expression (P<0.05, Figure 3).

Figure 3 CCL5 knockdown inhibits colon cancer cell proliferation, migration, and invasion in vitro. (A) Schematic representation of the lentiviral vector for human CCL5 knockdown. (B) qRT-PCR validation of CCL5 mRNA expression in CW-2 and HT-29 cells after lentiviral infection. (C) Representative images of scratch wound healing assay at 0 and 48 hours (crystal violet staining, ×40) and quantitative analysis of wound closure rate. (D) Colony formation assay and quantitative analysis of colony numbers (crystal violet staining). (E) CCK-8 cell viability assay showing proliferative capacity of CW-2 and HT-29 cells over 3 days. (F) Transwell migration assay (crystal violet staining, ×40) and quantitative analysis of migrated cells. (G) Transwell invasion assay (Matrigel-coated; crystal violet staining, ×40) and quantitative analysis of invaded cells. Data are presented as mean ± SD. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. CCK-8, Cell Counting Kit-8; KD, knockdown; NC, negative control; OD, optical density; qRT-PCR, quantitative reverse transcription polymerase chain reaction; SD, standard deviation.

Results of transcriptomics sequencing

Differently expressed genes

To determine which genes were differentially expressed between the control cell lines and the stably transfected cells overexpressing the CCL5 gene, RKO stably transfected cells and the control cells were prepared for transcriptome sequencing. A total of 227 DEGs were found in the two cell groups after modifying the threshold screening of P<0.05 and |log₂FC| >1.0. R was used to depict the results as heatmaps and volcano plots. Figure 4A,4B display the findings.

Figure 4 CCL5 overexpression alters the transcriptome and activates key signaling pathways in colon cancer cells. (A) Heatmap showing DEGs between RKO cells overexpressing CCL5 and control cells (|log₂FC| >1, P<0.05). (B) Volcano plot illustrating the distribution of DEGs, with red dots indicating upregulated genes and blue dots indicating downregulated genes. (C) Bubble plot of GO enrichment analysis showing the top enriched BPs, CCs, and MFs of DEGs. (D) Network diagram of GO enrichment analysis illustrating the relationships between enriched terms and genes. (E) Bubble plot of KEGG pathway enrichment analysis showing the top enriched pathways, including PI3K/AKT signaling pathway. (F) Network diagram of KEGG enrichment analysis depicting the interactions between enriched pathways and associated genes. BP, biological process; CC, component cellular; COVID-19, coronavirus disease 2019; DEGs, differently expressed genes; ECM, extracellular matrix; FC, fold change; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; MF, molecular function; OE, overexpression.

Findings from the enrichment analysis of DEGs

We used the cluster Profiler package in R software to assess the GO enrichment functions of DEGs, including molecular function (MF), biological process (BP), and cellular component (CC), in order to comprehend the functions of DEGs between the two groups of CC cancer cells following overexpression of the CCL5 gene. The cluster Profiler package, which includes MF, BP, and CC, was used to assess the GO enrichment function of genes that were differentially expressed. Following the P<0.05 filter adjustment, the following outcomes were attained: Response to virus, defense response to virus, and defense response to symbionts are the primary components of BP function; centromere, outer plasma membrane, and early endoplasmic body are the primary components of CC function; and GPCR binding, metallopeptidase activation, cytokine receptor binding, and metalloendopeptidase activation are the primary components of MF function. Figure 4C,4D display the findings.

We used the cluster Profiler package in the R software to analyze the KEGG enrichment function of DEGs in order to determine the downstream pathways of CCL5 gene action in CC cells and to comprehend the enrichment pathways of DEGs between the two groups of cells. After adjusting the P<0.05 screening, we obtained the following results: Human papillomavirus infection, coronavirus disease 2019 (COVID-19) virus, and activation of the PI3K/AKT signaling pathway. Figure 4E,4F display the findings.

CCL5 promotes tumor growth by triggering the PI3K/AKT signaling

According to the transcriptome sequencing results, PI3K/AKT signaling may be present in the pathway that is activated downstream of transfected cell lines that overexpress CCL5. One particular PI3K inhibitor is LY294002. The concentration at which LY294002 acted in this investigation was 10 µM. The outcomes of CCK-8 tests were used to assess the duration of activity in several stably transfected cell lines. The experimental findings demonstrated that the OD value was significantly lower than that of the control cell line, P<0.05, 18 hours after LY294002 was added to RKO stably transfected cells overexpressing the CCL5 gene; the OD value was significantly lower than that of the control cell line, P<0.05, 16 hours after LY294002 was added to SW480 stably transfected cells overexpressing the CCL5 gene. In this investigation, LY294002’s following time of action was 8 hours for RKO stable transplant cells and 6 hours for SW480 stable transplant cells. Figure 5A displays the findings. Furthermore, we conducted the Transwell tumor cell migration and invasion experiment, scratch assay, and plate clone formation assay. According to the experimental findings, LY294002 could (P<0.05) suppress the proliferative activity of RKO and SW480 cells that overexpress CCL5 (Figure 5B). LY294002 may prevent CCL5 overexpressing RKO and SW480 cells from migrating and invading (P<0.05) (Figure 5C-5E). According to these experimental findings, CCL5 activates the PI3K/AKT signaling pathway, which has a pro-tumorigenic effect. In this investigation, LY294002’s following time of action was 18 hours for RKO stable transplant cells and 6 hours for SW480 stable transplant cells. Figure 5A displays the findings. Furthermore, we conducted the Transwell tumor cell migration and invasion experiment, scratch assay, and plate clone formation assay. According to the experimental findings, LY294002 could suppress the proliferative activity of RKO and SW480 cells that overexpress CCL5 (P<0.05, Figure 5B). LY294002 may prevent CCL5 overexpressing RKO and SW480 cells from migrating and invading (P<0.05, Figure 5C-5E). According to these experimental findings, CCL5 activates the PI3K/AKT signaling pathway, which has a pro-tumorigenic effect.

Figure 5 PI3K/AKT inhibitor LY294002 reverses CCL5 overexpression-induced proliferation, migration, and invasion in colon cancer cells. (A) Time-course analysis of LY294002 (10 µM) inhibitory effect on RKO and SW480 cells overexpressing CCL5, assessed by CCK-8 assay. (B) Colony formation assay and quantitative analysis showing that LY294002 treatment suppressed the clonogenic capacity of RKO and SW480 cells overexpressing CCL5 (crystal violet staining). (C) Representative images of scratch wound healing assay at 0 and 48 hours (crystal violet staining, ×40) and quantitative analysis of wound closure rate. (D) Transwell migration assay (crystal violet staining, ×40) and quantitative analysis of migrated cells. (E) Transwell invasion assay (Matrigel-coated; crystal violet staining, ×40) and quantitative analysis of invaded cells. Data are presented as mean ± SD. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. CCK-8, Cell Counting Kit-8; OD, optical density; OE, overepression; SD, standard deviation.

Similarly, in the stable knockdown cell lines, we employed 740Y-P as a PI3K activator at a concentration of 10 µM. We then treated the CCL5-knockdown stable cells with 740Y-P to evaluate its effects on tumor cell proliferation, migration, and invasion. The results demonstrated that 740Y-P treatment significantly restored the proliferative, migratory, and invasive capacities of CCL5-deficient cells (P<0.05, Figure 6).

Figure 6 PI3K/AKT agonist 740Y-P rescues the malignant phenotype of CCL5-knockdown colon cancer cells. (A) Time-course analysis of 740Y-P (10 µM) agonistic effect on CW-2 and HT-29 cells with CCL5 knockdown, assessed by CCK-8 assay. (B) Colony formation assay and quantitative analysis showing that 740Y-P treatment restored the clonogenic capacity of CCL5-deficient CW-2 and HT-29 cells (crystal violet staining). (C) Representative images of scratch wound healing assay at 0 and 48 hours (crystal violet staining, ×40) and quantitative analysis of wound closure rate. (D) Transwell migration assay (crystal violet staining, ×40) and quantitative analysis of migrated cells. (E) Transwell invasion assay (Matrigel-coated; crystal violet staining, ×40) and quantitative analysis of invaded cells. Data are presented as mean ± SD. *, P<0.05; **, P<0.01; ****, P<0.0001. CCK-8, Cell Counting Kit-8; KD, knockdown; OD, optical density; SD, standard deviation.

STAT1 regulates CCL5 expression

PLK1, STAT1, and HSP90AA1 were identified as the key genes that might serve as the hub genes in the CCL5 overexpressing stable transplants by examining the protein interaction network of DEGs in overexpressing CCL5 cell lines. These are the genes that may be linked to the variations of CCL5 expression. Figure 7A displays the experimental results. The study used the KnockTF, hTFtarget, CHEA, and ENCODE databases to identify the possible transcription factors in CC patients in order to better understand the putative upstream regulatory genes of CCL5. Additionally, the TCGA database’s CC patient dataset served as the source of information for the correlation study. These included 262 transcription factors predicted by the TCGA database, 125 transcription factors predicted by the KnockTF database, 98 transcription factors predicted by the hTFtarget database, 13 transcription factors predicted by the CHEA. In each of the last five databases, a positive prediction for STAT3 was obtained by taking the intersection of the predicted transcription factors from these databases. Figure 7B displays the outcomes of the experiment. Given that the hub gene was anticipated to be STAT1 by the earlier PPI results of this study and that the CHEA database itself predicted fewer transcription factors. The re-analysis’s findings showed that STAT1 is a predicted transcription factor for CCL5 in all four databases, with the exception of the CHEA database. Therefore, using a dual luciferase reporter experiment, we constructed a plasmid overexpressing STAT1 in order to evaluate it as an upstream transcription factor of CCL5. Figure 7C displays the outcomes of the experiment. An additional RT-qPCR study showed that STAT1 is a CCL5 negative regulator. Figure 7D displays the outcomes of the experiment.

Figure 7 STAT1-mediated transcriptional repression of CCL5 inhibits tumor growth and modulates the PI3K/AKT pathway. (A) Transcriptome analysis of DEGs-protein interactions following CCL5 overexpression; (B) upstream transcription factors of CCL5 were predicted by several databases; (C) results of the dual luciferase reporting experiment; (D) alterations in STAT1 and CCL5 expression following STAT1 gene overexpression, indicating that STAT1 functions as a CCL5 negative regulator; (E) verification of the effectiveness of CCL5 overexpression in CT-26 cells; (F) data quantification findings and changes in mouse tumor volume following overexpression of the CCL5 gene; (G) data quantification results following in vitro CCL5 knockdown. **, P<0.01; ****, P<0.0001; ns, not significant. DEGs, differentially expressed genes; KD, knockdown; NC, negative control; OE, overexpression.

Animal study

We used lentivirus that overexpressed the mouse CCL5 gene and EG to infect mouse CC cells CT-26. Figure 7E displays the results of the expression verification. In the meantime, lentiviruses that carried the CCL5 gene knockdown and EG were used to infect HT-29 cells. Subcutaneous injections were administered to naked mice, and the development of subcutaneous tumors in the mice was monitored. Up-regulating CCL5 expression in vivo was shown to promote tumor development and proliferation, while down-regulating CCL5 expression was found to do the opposite. The findings are displayed in Figure 7F,7G.

CC cells with high CCL5 chemokine expression exhibit enhanced susceptibility to immune cell-mediated killing

Human peripheral blood lymphocytes (PBMCs) and CCL5-high-expressing CC cells were co-cultured, and it was discovered that the lymphocytes showed increased cytotoxic sensitivity to the tumor cells that expressed CCL5. In contrast, PBMCs were less effective in killing cancer cells when CCL5 expression was lower. These findings imply that the activation of PBMCs may be influenced by CCL5 expression levels. The findings are displayed in Figure S1.

Discussion

The key finding of this study is that CCL5 exerts pro-tumorigenic effects in CC via PI3K/AKT pathway activation, and its transcription is suppressed by STAT1. By releasing the killing potential of T cells, immunotherapies such as immune checkpoint inhibitors, T cells expressing chimeric antigen receptors, and tumor vaccines have significantly improved the treatment of cancer (5). Since CCL5 is a chemokine that attracts natural and particular immunological signaling-associated immune cells into tumors, its function in promoting the interaction between immune cells and malignancies has been well studied. The current study examined the functional effects of CCL5 expression on CC cells by creating stably-transformed CC cell lines that overexpress and knock down the CCL5 gene. This was done because there are fewer studies on the role of CCL5 itself on tumors and the mechanism is still unclear in CC patients. We discovered through both in vitro and in vivo investigations that the biological behavior of CC cells was greatly impacted by the level of CCL5 expression. The in vivo tests in this investigation used thymus-free nude mice since the chemotactic effect of CCL5 on immune cells may influence tumor genesis and growth. The ability of CC cells to proliferate, migrate, and invade was found to be considerably enhanced by higher CCL5 expression, while the opposite was true for lower CCL5 expression. This was verified by both in vitro and in vivo investigations. This implies that CCL5 itself may have a function in accelerating the development of CC. This clearly contradicts the results of our investigation of CCL5 expression and CC patient survival. The overall survival time of CC patients with high CCL5 expression was considerably longer than that of CC patients with low CCL5 expression, according to an analysis of the connection between CCL5 expression and overall survival time of CC patients using Kaplan-Meier plotter data. In this investigation, we examined the potential causes: As a chemokine, CCL5 has strong chemo attractive effects on immune cells, including mast cells, basophils, NK cells, T cells, dendritic cells, and eosinophils (22). Our findings also revealed that CC cells overexpressing CCL5 could significantly activate PBMCs, thereby enhancing their tumor-killing effects. This suggests that CCL5 may exert its tumor-suppressive function by modulating the immune microenvironment. However, further extensive experiments are required to elucidate the precise mechanisms through which CCL5 activates PBMCs and inhibits tumor initiation and progression.

Similar to other earlier research where CCL5 exerted the same pro-tumorigenic effects in different malignancies, we discovered that CCL5 enhances the proliferation, migration, and invasion of CC cells in ex vivo assays. According to earlier research, the tumor-associated fibroblast-derived chemokine CCL5 in hepatocellular carcinoma promotes metastasis in the mice’s lungs and increases the migration and invasion of hepatocellular carcinoma cells via activating the HIF1α/ZEB1 axis (23). Similarly, in gastric cancer, KLF5 was reported to increase the invasive and metastatic capacity of tumor cells by activating the CCL5/CCR5 axis in gastric cancer-associated fibroblasts (24). According to research on ovarian cancer, CCL5 is a potential effector molecule for miRNA regulation in tumor-associated fibroblasts, which aids in the recruitment and proliferation of tumor cells (25). Patients with advanced metastatic ovarian cancer had high levels of CCL5 in their serum (26). Research on breast cancer has demonstrated that the paracrine action of CCL5, which is released by tumor mesenchymal stem cells, promotes tumor cell motility, invasion, and metastasis (27). According to a substantial amount of clinical data, patients with melanoma, breast, cervical, prostate, gastric, and pancreatic cancers may have a poor prognosis if their tissue or plasma CCL5 levels are increased (28-34). Mounting clinical evidence demonstrates significant correlations between CCL5 gene expression and distinct molecular characteristics, gene expression profiles, TME immune cell infiltration patterns, and therapeutic benefits in colorectal cancer (CRC) (35). These findings suggest that targeting the CCR5/CCL5 axis may hold clinical promise for selected CRC subtypes and could play a pivotal role in developing innovative strategies to modulate the immunotherapeutic landscape of CRC. Regarding immunosuppressive mechanisms, studies have revealed that epidermal growth factor receptor signaling inhibition paradoxically upregulates CCL5 expression, subsequently promoting the recruitment of immunosuppressive cells, including tumor-associated macrophages and myeloid-derived suppressor cells, while also inducing immunoinhibitory polarization of macrophages within the TME (36). In animal investigations, it was also discovered that aniline, a novel CCR5 antagonist, inhibited the invasive and metastatic characteristics of prostate cancer cells in mice (37). In CRC xenograft models, anti-CCL5 neutralizing antibody treatment significantly attenuated tumor growth and reduced peritoneal and hepatic metastases (38). These observations in various cancers imply that CCL5 might have a significant pro-carcinogenic function in cancer. For the treatment of these cancers, CCL5 might be a crucial target.

Using transcriptome sequencing of CC cells that were stably transfected to overexpress the CCL5 gene, we discovered that the pathway downstream of CCL5 is PI3K/AKT pathway. This is in line with earlier research. In various disorders, it has been discovered that downstream pathways, such as PI3K/AKT and NF-κB pathways, are linked to inflammation, invasion, division, apoptosis, angiogenesis, and cell proliferation (39,40). One important signaling cascade implicated in several physiological and pathological states is the PI3K/AKT pathway (41). Numerous malignancies are caused by the PI3K/AKT pathway, which controls the translation of mRNA encoding precancerous proteins. Resulting in the survival of malignant cells in a variety of malignancies. By changing several genes that may impact the biological functions of tumor cells, this pathway is crucial in controlling tumor invasion, migration, and proliferation (42,43). A theoretical foundation for the eventual development of possible target therapeutic drugs was established by this work, which demonstrated that the overexpression of CCL5 was linked to the activation of the PI3K/AKT signaling pathway. Furthermore, using bioinformatics, this study discovered that STAT1 might be an upstream regulator of CCL5. The dual luciferase reporter assay verified the reciprocal relationship between STAT1 and CCL5, and the reduction in CCL5 expression following STAT1 overexpression suggested that STAT1 is a negative regulator of CCL5. This is in line with earlier research showing that STAT1 regulates cytokines (44). The current study demonstrated the negative regulatory role of STAT1 on CCL5 of CCL5, which may provide a potential target for the subsequent treatment of CC patients. Previous research has even demonstrated that STAT1 can be identified as a predictor of immunotherapy (45), indicating that STAT1 plays an important role in immune regulation.

An intriguing observation from our clinical data is that CCL5 expression was lower in malignant tissues compared to paracancerous tissues (Figure 1B), which appears to contradict our experimental findings that CCL5 promotes tumor cell proliferation, migration, and invasion. This paradox may be explained by the dual, context-dependent roles of CCL5 in CC. While tumor-cell-intrinsic CCL5 signaling directly drives malignant behaviors via the PI3K/AKT pathway, its potent chemotactic function in the TME recruits anti-tumor immune effectors. Therefore, established tumors may undergo immune editing, selectively favoring clones with reduced CCL5 expression to evade immune attack, resulting in an overall lower average in tumor bulk. This hypothesis is supported by our finding that, among tumor tissues themselves, higher CCL5 levels correlate with better differentiation and improved patient survival (Figure 1D, Table 1), likely reflecting a more immunogenic tumor phenotype. Thus, the net expression level in clinical samples represents a balance between the pro-tumorigenic autocrine signal and the anti-tumor immune pressure it elicits.

While our study provides novel insights, several limitations should be acknowledged: First, we observed an intriguing clinical paradox where higher CCL5 expression correlated with better prognosis in CC patients, despite our experimental findings demonstrating CCL5’s pro-tumorigenic effects in promoting proliferation, migration and invasion. This discrepancy suggests that CCL5 may exert context-dependent, dual roles in CC pathogenesis through yet unidentified tumor-suppressive mechanisms. Our current study has not fully elucidated these potential protective functions of CCL5, which warrants further investigation. Second, although we focused on the PI3K/AKT pathway as a key mediator of CCL5’s oncogenic effects, other signaling pathways may contribute to its complex biological functions. Future studies should systematically explore additional mechanisms, particularly those that might explain CCL5’s apparent tumor-suppressive effects in clinical observations. To sum up, our findings showed that CCL5 markedly increased CC cell invasion, migration, and proliferation both in vivo and ex vivo. This implies that for CC patients, CCL5 is a trustworthy possible treatment target. It is necessary to weigh the tumor-promoting properties of CCL5 against drug studies that use it as an immunotherapeutic target.

Conclusions

In CC tissues, CCL5 expression varies from normal intestinal tissues to malignant and paracancerous tissues. Future research is still required to determine whether CCL5 and the degree of differentiation of CC are favorably connected. By activating the PI3K/AKT signaling pathway, CCL5 has a pro-tumorigenic effect and encourages the growth, migration, and invasion of CC cells both in vivo and ex vivo. Furthermore, the transcription factor STAT1 negatively impacts CCL5 expression.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR and ARRIVE reporting checklists. Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2025-aw-917/rc

Data Sharing Statement: Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2025-aw-917/dss

Peer Review File: Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2025-aw-917/prf

Funding: This study was supported by the Yunnan Province High-level Talent Training Support Plan-Special Project for “Famous Doctors” (No. RLMY20200019), the Doctoral Students Innovation Fund (No. 2024B022) and the Provincial Basic Research Plan (No. 2018FE001-035).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jgo.amegroups.com/article/view/10.21037/jgo-2025-aw-917/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. The study was approved by the Ethical Committee of The First Affiliated Hospital of Kunming Medical University [IRB: (2020) Ethics L No. 159; approval date: March 8, 2024]. Verbal informed consent was obtained from all participants or their family members via telephone prior to the use of their archived tissue specimens for research. All animal experiments were performed under a project license (No. kmmu20240669) granted by the Animal Ethics Committee of Kunming Medical University, in compliance with institutional guidelines for the care and use of animals.

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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