The impact and mechanisms of CRIP2 on the biological behavior of triple-negative breast cancer cells

Introduction

In recent years, the incidence rates of breast cancer have been among the highest for malignant tumors in women, posing a severe threat to women’s health (1,2). Triple-negative breast cancer (TNBC) is widely regarded as the most aggressive subtype of breast cancer.

Cysteine-rich intestinal protein 2 (CRIP2) is a member of the LIM domain protein family, The LIM protein family usually affects biological functions by mediating protein-protein interactions (3), and the wide distribution and high level of expression of CRIP2 in different tissues suggest that CRIP2 plays an important role in cellular functions (4-6). In recent years, with the in-depth study of copper ion-related cell death modes, CRIP2 has been shown to mediate copper metabolism as a copper ion-binding protein (7). In the field of tumor research, CRIP2 has been shown to act as an autophagy inhibitor to regulate the activation of autophagy in non-small cell lung cancer (NSCLC) cells by mediating copper metabolism (8). And furthermore research has shown that CRIP2 can induce apoptosis in esophageal cancer cells and inhibit the progression of esophageal cancer (9). In a study of cutaneous squamous cell carcinoma, it was demonstrated that the interaction between HOXA9 and CRIP2 at the promoter of the glycolysis gene blocked the binding of hypoxia-inducible factor 1α (HIF-1α) and inhibited the expression of the gene in trans. This, in turn, acted as a tumour suppressor and inhibited glycolysis in cutaneous squamous cell carcinoma (10). Furthermore, it acts as a target gene for miR-449a, influencing the migration and invasion of TNBC cells (11). However, our comparison using The Cancer Genome Atlas (TCGA) database revealed that CRIP2 expression is higher in breast cancer tissues than in normal breast tissues, which contradicts previous studies. Therefore, this study aims to investigate the role and mechanism of CRIP2 in TNBC cells, specifically in the MDA-MB-231 cell line.

Mitogen-activated protein kinase kinase 4 (MAP2K4) is a crucial member of the mitogen-activated protein kinase activator family (12). Our previous findings indicated that MAP2K4 exhibits oncogenic properties in breast cancer by interacting with vimentin to activate the PI3K/AKT pathway, thereby enhancing the proliferation, migration, and invasion of breast cancer cells (13). Concurrent with the course of our other study, we ascertained that MAP2K4 could regulate the expression of CRIP2 protein level. Thus, the present study was a preliminary investigation of the relationship between MAP2K4 and CRIP2. We present this article in accordance with the MDAR reporting checklist (available at https://tbcr.amegroups.com/article/view/10.21037/tbcr-24-57/rc).

Methods

Cell sources

MCF-10A (normal breast epithelial cells) were purchased from Pricella Biotechnology (Pricella, Wuhan, China). MCF-7 (luminal type breast cancer cells), MDA-MB-231 (TNBC cells), and SKBR3 (HER-2 positive breast cancer cells) were gifted by the Central Laboratory of Integrated Chinese and Western Medicine Hospital, Southern Medical University. In subsequent functional experiments, MDA-MB-231 cells were divided into an overexpressing CRIP2 group (OE-CRIP2) and a negative control (NC) group. The cells were transiently transfecting with an overexpressing CRIP2 plasmid versus a NC plasmid that had no effect on CRIP2 expression.

Primary instruments and materials

Primary equipment

Cell incubators, −80 ℃ freezers, biosafety cabinets, and multi-wavelength microplate readers (ThermoFisher Scientific); protein vertical electrophoresis and transfer system (Bio-Rad); immunoblot imaging system (MiniChemi); ultrapure water systems (Millipore); inverted and upright fluorescence microscopes (Olympus).

Primary materials

DMEM medium, 0.25% trypsin, and Opti-MEM low serum medium (Gibco); Lipofectamine 3000 (ThermoFisher Scientific); fetal bovine serum (Zeta Life); protein electrophoresis gels, pre-stained protein markers, and enhanced chemiluminescence (ECL) reagent (Yeasen); RIPA lysis buffer, protease inhibitors, and phosphatase inhibitors (KangWei Century); EdU kit (RiboBio); Cell Counting Kit-8 (CCK-8) cell proliferation and cytotoxicity assay (Biosharp); Transwell chambers (Corning); MAP2K4 overexpression lentivirus and CRIP2 overexpression plasmid (Genechem). Primary antibodies for MAP2K4 and CRIP2 (Proteintech); P65 and p-P65 antibodies (CST); and internal control and secondary antibodies (Bioworld).

Study methods

Cell culture

MCF-10A cells were cultured using the proprietary medium provided by Precella Biotechnology. MCF-7, SKBR3, and MDA-MB-231 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) medium containing 10% fetal bovine serum. All cell lines were maintained in a humidified incubator at 37 ℃ with 5% CO₂.

Bioinformatics analysis of CRIP2 expression in breast cancer and normal tissues

The expression differences of CRIP2 in breast cancer and normal tissues were analyzed using bioinformatics tools. The UALCAN database (https://ualcan.path.uab.edu/index.html) and the GEPIA database (http://gepia.cancer-pku.cn/) were used to retrieve TCGA and GTEx data to compare the expression of CRIP2 in breast cancer and adjacent normal tissues. While UALCAN contains only the BRCA dataset from the TCGA database (normal n=114; primary tumor n=1,097), GEPIA contains the BRCA dataset from the TCGA database and the normal breast tissue dataset from the GTEx database (normal n=291; tumor n=1,085) (14).

Cell transfection

CRIP2 overexpression plasmids and MAP2K4 overexpression lentivirus were obtained from Genechem Biosciences (Shanghai, China). MDA-MB-231 cells were plated into a six-well plates at 30–50% confluence, 24h before transfection. Plasmids were transfected into MDA-MB-231 cells at a final concentration of 50 nmol/L using Lipofectamine 3000 (Invitrogen, Guangzhou, China) in serum-free conditions. Evaluate after 8–12 hours of transfection, and if the cells appear to be in good condition (morphology, wall adherence, etc.), extend the transfection time to 24 hours. 8–12 (or 24) hours later, the medium was replaced by DMEM. When the cell confluence reaches 80–90%, extract the cells for functional assay or extract the protein for Western blot (WB). MAP2K4 lentiviral transfection was performed as described in the citation (13). Transfection efficiency verified by WB.

CCK-8 cell proliferation assay

MDA-MB-231 cells were dispensed into a 96-well plate at a cell density of approximately 1,000–2,000 cells per cell, purchased from Sorfa Life Science Research (Zhejiang, China). After cell attachment, cell viability was assessed using the Cell Counting Kit-8 (CCK-8; Biosharp, Hefei, China) at predetermined times of 0, 24, 48 and 72 hours. Briefly, cells in each well were incubated with 10 µL of CCK-8 solution for 2 hours. Absorbance was then measured at 450 nm using an enzyme-linked immunosorbent assay.

EdU cell proliferation assay

MDA-MB-231 cells proliferation was assessed using a Cell-Light EdU Apollo 567 In Vitro Imaging Kit (RiboBio, Guangzhou, China) according to the manufacturer’s guidelines. Briefly, cells were incubated with 10 mM EdU for 2 hours, and then fixed with 4% paraformaldehyde solution. The cells were then permeabilized with 0.5% Triton X-100 and stained with Apollo and Hochest fluorescent dyes for 30 minutes. The number of EdU-positive cells was determined using a fluorescence microscope. This procedure was performed three times independently for validation.

Transwell migration and invasion assay

Cells were transferred and assessed using Transwell chambers (purchased from Corning, NY, USA). The transwell chambers in 24-well plates were coated with or without Matrigel (diluted 1:6, obtained from BD Biosciences). Specifically, 1×10⁵ MDA-MB-231 cells immersed in serum-free medium were placed in the upper chamber, while complete medium supplemented with 20% FBS was placed in the lower chamber. After the incubation period of 12 h, the upper surface of the upper chambers was carefully wiped with cotton-tipped applicators. Migrated cells on the bottom were fixed with paraformaldehyde for 20 minutes. This step was followed by staining with Giemsa stain kit (#D011-2-3, obtained from Jiancheng bioengineer, Nanjing, China) for 20 minutes (for the specific staining method, please refer to the kit manual). After staining, the cells were washed and imaged under an inverted microscope.

Protein extraction and Western blotting

Proteins were extracted from cells using RIPA lysis buffer from Cwbiotech (Taizhou, China) supplemented with phosphatase and protease inhibitors (100:1:1). Protein concentrations were quantified using a BCA protein assay kit from Thermo Scientific (ThermoFisher Scientific, MA, USA). Proteins were then separated on a 10% SDS-PAGE gel and transferred to PVDF membranes supplied by Millipore (ThermoFisher Scientific, MA, USA). The membranes were blocked with nonfat milk for 1 hour and stained with anti-CRIP2 (Proteintech, 14801-1-AP, 1:500), anti-MAP2K4 (Proteintech, 67333-1-Ig, 1:5,000), anti-phospho-NF-κB p65 (Ser536) (CST, #3031, 1:1,000), and anti-GAPDH (Bioworld, MB66349, 1:15,000) antibodies overnight at 4 ℃. The next day, the membranes were washed three times with TBST and then incubated for 2 hours with goat anti-rabbit/mouse IgG (H&L) highly cross-adsorbed with HRP (Bioworld, BS22357/BS22356, 1:5,000). Antigen-antibody interactions were visualized using enhanced chemiluminescence reagents from Epizyme Biotech (Shanghai, China).

Statistical analysis

Statistical analysis was performed using SPSS 27. For normally distributed samples, Student’s t-test was used to compare two independent samples. Analysis of variance (ANOVA) was used for multiple group comparisons. A P value <0.05 was considered statistically significant, represented in figures as *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, and ns (not significant).

Results

Tumour/normal differential expression analysis confirms higher CRIP2 expression in breast cancer tissues than in normal tissues

Using two online bioinformatics platforms, we analysed the differential expression of CRIP2 in breast cancer and normal breast tissue datasets from different databases. The UALCAN database, by comparing the BRCA dataset in TCGA (normal n=114; primary tumour n=1,097), showed that the overall expression of CRIP2 was higher in breast cancer tissues than in normal tissues, as shown in Figure 1A. Due to the small sample size of normal breast tissues in the TCGA dataset, and to avoid possible errors due to small sample size, we also used the GEPIA database, which contains both the TCGA and GTEx datasets, to compare the differential expression of CRIP2 (normal n=291; tumour n=1,085). The results showed that CRIP2 expression was significantly higher in breast cancer tissues than in normal tissues, as shown in Figure 1B (all abbreviations on the horizontal axis represent different cancer abbreviations, where BRCA stands for breast cancer). Both databases showed that CRIP2 expression was higher in breast cancer tissue than in normal breast tissue.

Figure 1 Tumour/normal differential expression analysis confirms higher CRIP2 expression in breast cancer tissues than in normal tissues. (A) Differential expression of CRIP2 in the UALCAN database (TCGA-BRCA dataset, normal n=114; primary tumour n=1,097). (B) Differential expression of CRIP2 in different cancer types in the GEPIA database (BRCA stands for breast invasive carcinoma dataset, normal n=291; tumour n=1,085). CRIP2, cysteine-rich intestinal protein 2; TCGA, The Cancer Genome Atlas.

Low expression of CRIP2 in TNBC cells

To investigate the expression of CRIP2 in TNBC, we analyzed the expression profile of CRIP2 in breast cancer cells using the CCLE database. The results indicated that CRIP2 expression was lower in common TNBC cell lines compared to other subtypes, which significantly differed from the bioinformatics analysis results shown in Figure 1, as illustrated in Figure 2A (common TNBC cells are represented by the red squares). We hypothesized that this discrepancy might be due to the high heterogeneity of TNBC. To confirm this hypothesis, we verified the expression profile of CRIP2 in different breast cancer cell subtypes through Western blotting. The results demonstrated that CRIP2 expression was lower in MDA-MB-231 cells compared to MCF-10, SKBR3, and MCF-7 cells, as shown in Figure 2B. CRIP2 expression is lower in TNBC cells than in the other two subtypes of breast cancer cells.

Figure 2 Expression profile of CRIP2 in breast cancer cells. (A) Expression of CRIP2 in breast cancer cells from the CCLE database (common triple-negative breast cancer cells are represented by the red squares). (B) Protein band quantification and statistical analysis of CRIP2 expression levels across different cell lines (*P<0.05, ***P<0.001, n=3). CRIP2, cysteine-rich intestinal protein 2.

The impact of CRIP2 on the biological functions of MDA-MB-231 cells

In order to investigate the effect of CRIP2 on the biological function of MDA-MB-231 cells, a series of experiments were conducted. Firstly, MDA-MB-231 cells overexpressing CRIP2 were constructed by transient transfection, and the transfection efficiency was examined by WB (Figure 3A). Secondly, a CCK-8 assay, an EdU assay, and a Transwell invasion and migration assay were performed. The results of the CCK-8 proliferation assay indicated that compared to the NC group, the over-expression CRIP2 group (OE-CRIP2) significantly reduced the proliferation activity of MDA-MB-231 cells in a time-dependent manner, as shown in Figure 3B. The EdU proliferation assay further demonstrated that the number of proliferating cells in the OE-CRIP2 group was significantly lower than that in the NC group, as depicted in Figure 3C. Additionally, the Transwell migration and invasion assays revealed that upregulation of CRIP2 diminished the migratory and invasive capacities of MDA-MB-231 cells, as illustrated in Figure 3D. Overexpression of CRIP2 inhibits proliferation, migration and invasion of MDA-MB-231 cells.

Figure 3 Impact of CRIP2 on biological functions of MDA-MB-231 cells. (A) WB assay for OE-CRIP2 transfection efficiency (***P<0.001, n=3). (B) The results and statistical analysis of the CCK-8 proliferation assay (***P<0.001, ****P<0.0001, n=3). (C) The results and statistical analysis of the EdU proliferation assay (**P<0.01, n=3, scale bar 200 μm). (D) The results and statistical analysis of the Transwell migration/invasion assay (**P<0.01, ***P<0.001, n=3, scale bar 200 μm; staining with Giemsa stain kit). CCK-8, Cell Counting Kit-8; CRIP2, cysteine-rich intestinal protein 2; EdU, 5-ethynyl-2'-deoxyuridine; NC, negative control; OD, optical density; OE, overexpression; WB, Western blot.

CRIP2 influences the NF-κB pathway

The Biogrid database (https://thebiogrid.org/) has been identified as a potential resource for the analysis of protein-protein interactions. The database grid map under consideration suggests the possibility that interactions between two genes may have been previously reported or predicted by high-throughput sequencing data. Specifically, the analysis identifies Recombinant V-Rel Reticuloendotheliosis Viral Oncogene Homolog A (RelA, also known as P65) as a potential interacting protein with CRIP2, as shown in Figure 4A. A review of the pertinent literature revealed that CRIP2 has been demonstrated to interact with P65, thereby impeding the expression of P65 in the presence of pro-angiogenic cytokines, such as interleukin (IL)-6, IL-8, and vascular endothelial growth factor (VEGF). This results in the exertion of an inhibitory effect on tumour formation and angiogenesis (15). In summary, we hypothesised that the function of CRIP2 in the inhibition of the biological function of MDA-MB-231 cells might be associated with NF-κB P65. Therefore, we examined the effect of CRIP2 on the protein expression of P65 versus p-P65. The results demonstrated that the over-expression of CRIP2 inhibited the phosphorylation of P65, but did not affect the total protein expression of P65, as illustrated in Figure 4B. Previous studies have shown that CRIP2 interacts with P65, and our WB confirms that CRIP2 affects the phosphorylation of P65, which may underlie the molecular pathway by which CRIP2 exerts its oncogenic effects.

Figure 4 The influence of CRIP2 on the NF-κB pathway. (A) Biogrid database predicts RELA (NF-κB P65) as an interacting protein of CRIP2, as marked by the red circle. (B) Bands and statistical analysis of P65 and p-P65 proteins (***P<0.001, ****P<0.0001, n=3). CRIP2, cysteine-rich intestinal protein 2; NC, negative control; OE, overexpression.

CRIP2 regulates the biological functions of MAP2K4

In the preliminary experiment, it was established that the over-expression of MAP2K4 led to a decrease in CRIP2 protein expression. This finding was subsequently validated through a second experiment, which yielded consistent results (see Figure 5A). In our previous experiments, we demonstrated that the over-expression of MAP2K4 promoted the proliferation, migration and invasive ability of breast cancer cells, including the MDA-MB-231 cell line. In order to verify whether CRIP2 affects the ability of MAP2K4 to promote the malignant phenotype of breast cancer, we performed further functional experiments, including CCK-8, EdU, and Transwell migration and invasion tests. We sought to ascertain whether CRIP2 affects the ability of MAP2K4 to promote the malignant phenotype of breast cancer. To this end, we constructed MDA-MB-231 cells stably overexpressing MAP2K4 by lentiviral construction and transiently transfected a plasmid overexpressing CRIP2 to construct MDA-MB-231 cells overexpressing both MAP2K4 and CRIP2, and performed a series of functional experiments, including CCK-8 and Transwell migration and invasion. The results of these experiments showed that CRIP2 expression reversed the ability of MAP2K4 to promote the proliferation, migration and invasion of MDA-MB-231 cells (Figure 5B,5C). MAP2K4 regulates CRIP2 expression, and at the same time, CRIP2 can affect the ability of breast cancer cells to promote the malignant phenotype caused by MAP2K4 overexpression.

Figure 5 The impact of CRIP2 on the MAP2K4-mediated biological functions of breast cancer cells. (A) The CRIP2 protein bands and statistical results (**P<0.01, n=3). (B) The CCK-8 assay results and statistical analysis (**P<0.01; ***P<0.001; ****P<0.0001, n=3). (C) The Transwell assay results and statistical analysis (**P<0.01, ***P<0.001, ****P<0.0001, n=3, scale bar 200 μm; staining with Giemsa stain kit). CCK-8, Cell Counting Kit-8; CRIP2, cysteine-rich intestinal protein 2; MAP2K4, mitogen-activated protein kinase kinase 4; NC, negative control; OE, overexpression.

Discussion

TNBC, characterised by its aggressive clinical behaviour and lack of targeted therapeutic options beyond conventional chemotherapy, continues to have the poorest prognosis among breast cancer subtypes (16). Our study identifies CRIP2 as a potential molecular regulator in TNBC pathogenesis, with significantly reduced expression levels observed in TNBC cells compared to other breast cancer subtypes. Functional analyses showed that CRIP2 overexpression effectively suppressed the proliferative, migratory and invasive capacities of MDA-MB-231 cells in vitro. These findings are consistent with emerging research priorities in TNBC biology, particularly the exploration of novel therapeutic targets such as trophoblast cell surface antigen 2 (TROP-2), which has recently gained prominence through its demonstrated ability to modulate the PI3K/AKT signalling axis (17-20).

Building on our previous discovery that MAP2K4 regulates PI3K/AKT signalling in breast cancer (13), the current investigation establishes CRIP2 as a downstream mediator of MAP2K4-driven oncogenic effects. Notably, MAP2K4 upregulation induced CRIP2 downregulation, whereas CRIP2 overexpression counteracted MAP2K4-mediated enhancement of malignant phenotypes. This regulatory interplay suggests that CRIP2 may serve as a critical checkpoint in the MAP2K4-PI3K/AKT signalling network, potentially explaining the dual involvement of this pathway in tumour progression and therapeutic resistance. Future studies will evaluate the therapeutic potential of combinatorial targeting strategies involving TROP2 inhibitors and MAP2K4/CRIP2 axis modulators.

Our mechanistic investigation further implicates NF-κB signalling in CRIP2-mediated tumour suppression. The observed reduction in phosphorylated P65 (p-P65) levels upon CRIP2 up-regulation—without concomitant changes in total P65 expression—suggests CRIP2-mediated modulation of NF-κB activation status. This finding extends previous reports of crosstalk between the PI3K/AKT and NF-κB pathways (21-23). We propose a novel regulatory mechanism by which CRIP2 may coordinate signalling convergence between these critical oncogenic pathways. Pharmacological inhibition experiments using helenalin, a selective P65 inhibitor, are planned to validate this hypothesis through systematic pathway analysis.

Several limitations must be considered when interpreting these findings. Current conclusions are constrained by technical limitations in CRIP2 antibody specificity, which preclude immunohistochemical validation in clinical specimens and chromatin immunoprecipitation assays to investigate potential MAP2K4-CRIP2 interactions. Furthermore, the focus of the study on MDA-MB-231 cells requires caution in generalising the results to TNBC subtypes or other breast cancer classifications. Future investigations will prioritise (I) multi-centre validation of CRIP2 expression patterns in annotated TNBC tissue arrays, (II) structural characterisation of MAP2K4-CRIP2 molecular interactions and (III) in vivo evaluation of the therapeutic potential of CRIP2 using orthotopic xenograft models.

Conclusions

Our findings demonstrate that CRIP2 functions as a tumor suppressor in TNBC through dual regulatory mechanisms: (I) direct suppression of malignant behaviors via NF-κB pathway modulation, evidenced by reduced phosphorylated P65 levels upon CRIP2 overexpression; (II) dynamic interplay with MAP2K4 signaling, where CRIP2 downregulation mediates MAP2K4-driven oncogenic effects while its restoration reverses these pro-tumorigenic phenotypes.

Acknowledgments

Our profound appreciation is directed towards the Central Laboratory of the Integrated Hospital of Traditional Chinese Medicine, Southern Medical University for generously availing the experimental facilities and extending technical expertise.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://tbcr.amegroups.com/article/view/10.21037/tbcr-24-57/rc

Data Sharing Statement: Available at https://tbcr.amegroups.com/article/view/10.21037/tbcr-24-57/dss

Peer Review File: Available at https://tbcr.amegroups.com/article/view/10.21037/tbcr-24-57/prf

Funding: This research venture was incidentally funded through a grant from the National Natural Science Foundation of China (grant No. 82060480) and the Doctoral Research Startup Fund of Guizhou Medical University, 2021 (GYFYBSKY-2021-42).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tbcr.amegroups.com/article/view/10.21037/tbcr-24-57/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

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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