Tumor suppressor gene chondroadherin opposes migration and proliferation in breast cancer and predicts a good survival.

Introduction
Breast cancer (BC) is the most frequently diagnosed cancer among women worldwide and a leading cause of cancer-related mortality. Various factors contribute to the risk of developing breast cancer, such as female sex, age ≥ 50 years, and family history [1]. Breast cancer is divided into several subtypes, which guide treatment: Hormone Receptor-Positive (HR+): This subtype includes cancers that are positive for estrogen or progesterone receptors. Treatment: Hormone therapies like Tamoxifen or Aromatase Inhibitors are used to block hormones that promote cancer growth. HER2-Positive: Cancers overexpressing the HER2 protein. Treatment: Targeted therapies such as Trastuzumab (Herceptin) are used to inhibit HER2-driven growth. Triple-Negative Breast Cancer (TNBC): Lacks estrogen, progesterone, and HER2 receptors. Treatment: Primarily chemotherapy, since these cancers do not respond to hormone or HER2-targeted therapies. Immunotherapy is under investigation. These treatments are tailored based on the specific characteristics of each subtype to enhance their effectiveness. Thus, prognostic biomarkers and potential therapeutic targets are urgently needed to improve the survival of BC patients and facilitate drug development.
In the past few years, efforts have been devoted to screening molecular biomarkers for predicting survival and are offered as potential therapeutic targets. For example, upregulated KI-67 [2] was found to be associated with poor prognosis, and resistance to chemotherapy. Negative regulators of the PI3K/AKT signaling pathway are correlated with good prognosis and oppose migration and proliferation [3]. Overexpression of EGFR is a negative indicator for survival in BC, and a similar trend was observed in another gene HER2 [4]. MicroRNAs associated with prognosis were also investigated in accordance with recent reports [5, 6]. Hormone therapies such as Tamoxifen and Aromatase Inhibitors (e.g., Anastrozole) are effective in treating ER-positive and PR-positive cancers by blocking hormone action or production. Targeted therapies like Trastuzumab (Herceptin) and Pertuzumab are used to inhibit HER2 signaling. PARP inhibitors, such as Olaparib, are particularly effective in treating BRCA mutation-associated cancers by exploiting the cancer’s DNA repair deficiencies. However, the currently used biomarkers for prognosis and potential drugs are still limited.
In this article, we report that another tumor suppressor gene, CHAD, is significantly lower in groups with high levels of malignancy, such as Triple-Negative Breast Cancer.
,Metastasis-prone Breast Cancer, Chemotherapy-resistant Breast Cancer, compared to low levels across several independent datasets [7, 8]. The clinical significance of CHAD, including its prognostic value, clinicopathological indicator association and expression was evaluated. CHAD can have a directly assessable inhibitory effect on tumor growth and can prevent metastasis [9, 10]. Functional assays were also performed to test the migration and proliferation rates following the knock-down of CHAD. KEGG pathways including “focal adhesion”, “ECM receptor interaction“ [11], “regulation of actin cytoskeleton” [12] and “PI3K/Akt pathway” were significantly enriched.

Materials and methods

Quantification of CHAD via RT-PCR
Total RNA was extracted from cancerous and normal tissues using TRIzol reagent (Invitrogen, CA, USA), according to the manufacturer’s instructions. RNA quantity and quality were assessed using a Nano Drop 2000 (Thermo Scientific, USA). First-strand cDNA was synthesized from 3 µg total RNA using random primers and M-MLV Reverse Transcriptase (Invitrogen, CA, USA). The relative expression of CHAD was measured by real-time PCR (RT-PCR) with an ABI PRISM 7900 sequence detector (Applied Biosystems, Carlsbad, CA, USA) and SYBR Green (Applied TaKaRa, Japan) according to the manufacturer’s protocols. The relative expression values were normalized to the endogenous control 18 S RNA and CT values. CHAD (Chondroadherin) Primers: Forward Primer: 5’-ATGGCACAGCCATCTACATC-3’, Reverse Primer: 5’-CTTGGTCTGGGATTTGCTCT-3’. 18 S RNA(used as a house keeping gene ) Primers: Forward Primer: 5’-CGGCTACCACATCCAAGGAA-3’, Reverse Primer: 5’-GCTGGAATTACCGCGGCT-3’. All samples were tested in duplicate and the mean values were retained for further analysis.

Cell culture and transfection
CHAD siRNA was obtained from Biomics Biotechnologies Co. (Shanghai, China), and the sense sequences were 5’- UGC AUC AUA GGU CGA CUA UTT − 3’ and 5’- AUA GUC GAC CUA UGA UGC ATT − 3’. Transfections were performed using INTERFERin reagent (Polyplus), as directed by the manufacturer. For each well (6-well plate, as an example), 11 µl (20 µM storage concentration) of siRNA duplexes was diluted in 200 µl of serum-free medium. After mixing, 12 µl of INTERFERin was added to the siRNA, vortexed, and incubated at room temperature for 10 min for complex formation. The mixture was then added to 2 ml of cell culture medium to achieve a final siRNA concentration of 100 nM, followed by gentle swirling of the plates. The incubation time for cells after siRNA transfection is 24 h. Overexpression plasmids for CHAD was obtained from Biomics Biotechnologies Co. (Shanghai, China). Seed cells in 6-well plates at a density of 2 × 10^5 cells per well one day prior to transfection. Transfect cells with the gene expression vector using Lipofectamine 2000/3000 according to the manufacturer’s protocol. Include an empty vector as a negative control.

Western blot analysis
Proteins were extracted using RIPA Lysis Buffer (Thermo Fisher Scientific), following the manufacturer’s instructions, and lysates were centrifuged (Thermo Fisher Scientific Centrifuge)at 12,000 rpm for 15 min. The protein concentration was determined using a bicinchoninic acid assay. CHAD antibody (ab204385, Abcam, Shanghai, China) was used at a dilution of 1:500, with Tubulin at 1:10,000 (ab15246, Abcam, Shanghai, China) and GAPDH at 1:10,000 (sc-47724,Santa Cruz Biotechnology) serving as the endogenous control. Immunocomplexes were incubated with a fluorescein-conjugated secondary antibody and signals were detected using an Odyssey infrared scanner (Li-CorBiosciences, Inc.). Preparation of the Invasion Chamber (Transwell):

Cell invasion assay
Use Transwell chambers with membranes having an 8 μm pore size. For a matrix gel invasion assay, pre-coat the membrane with a layer of Matrigel. Collect and count the cells. Suspend the cells in a serum-free medium. The cell density can vary from tens of thousands to hundreds of thousands per well, depending on the experimental purpose.Add a specific volume of the cell suspension (e.g., 100 µL) to the upper chamber of the Transwell.Add culture medium containing an attractant (e.g., medium with 10% FBS) to the lower chamber as an inducement signal for cell migration.Place the chamber in an incubator at 37 °C with 5% CO2 and incubate for 12 h. After incubation, gently wash the chamber with PBS to remove non-migrated cells.Fix the membrane with methanol or another appropriate fixative (usually for 10–15 min).Stain the membrane with crystal violet or another stain to enhance the visibility of migrated cells.Gently rinse to remove excess stain.Use a microscope to observe and photograph the membrane.Count the number of migrated cells in randomly selected fields of view, or perform quantitative analysis using image analysis software.Compile the counts from multiple fields, and calculate the average and standard deviation.

Migration and proliferation assays
The migration capacity of BC cell lines was assessed using Trans-well filter chambers (Costar, Corning, NY, USA) in accordance with the manufacturer’s protocols. Approximately 1 × 10^4 cells were suspended in serum-free medium and added to the top chamber, and 10% FBS medium, which refers to cell culture media that is supplemented with 10% fetal bovine serum (FBS), was placed in the bottom chamber. After h 12-hour incubation, cells that migrated to the lower membrane surface were stained, photographed, and counted under a microscope in three random fields per sample. All experiments were conducted in triplicates. For proliferation assays, BC cells were seeded into 96-well plates (3000 cells/well), and proliferation rates were measured every 24 h using a Cell Counting Kit-8 (Dojindo Laboratories, Japan) according to the manufacturer’s instructions.

Statistical analysis
Data analysis and visualization were performed using R and the relevant R packages. To analyze CHAD expression levels in a breast cancer-related dataset from the GEO database and compare them by breast cancer subtype using GraphPad for unpaired T-tests. Survival analysis utilized the “survival” package, and GO Enrichment analysis was conducted with the “clusterProfiler” package. Reference pathways for KEGG analyses were downloaded from 1000 permutations [13, 14]. The correlation between CHAD expression and clinical data was evaluated using Fisher’s exact test, with CHAD expression stratified into high and low groups based on median expression values.

Results

CHAD is down-regulated in BC tissues, especially in the groups with a high degree of malignancy
The expression values of CHAD in different pathological and clinical classification groups were compared in three independent datasets (Fig. 1A, D and E), including GEO datasets (GDS4069, N = 19), TCGA-BC(N = 1247), and CPTAC samples (N = 143; N = 108; The left graph includes 143 samples, consisting of 18 normal samples and 125 primary tumor samples. The right graph includes 108 samples, with 18 normal samples, 64 luminal samples, 10 HER2-positive samples, and 16 triple-negative samples.). PAM50 refers to a gene expression test that classifies breast cancer into intrinsic subtypes based on the expression profiles of 50 genes. This test helps categorize breast cancer into the following molecular subtypes: Luminal A, Luminal B, HER2-Enriched, Basal-like, Normal-like. The expression of CHAD was significantly lower in tumor tissues than in normal tissues and was most significantly reduced in the groups with a high degree of malignancy, such as triple-negative or basal-like breast cancerous tissues. ER-positive patients usually exhibit a lower degree of malignancy compared to ER-negative patients. As shown on the right side of Fig. 1D, the CHAD expression level is higher in the group of ER-positive patients. According to the THPA database, data from 62 different breast cancer cell line-related datasets showed that CHAD expression was relatively high in non-triple-negative cell lines (Fig. 1F). In addition to the aforementioned bioinformatics analysis results, experimental findings also demonstrate the same outcome. RT-PCR experiments on ten pairs of patient cancer and adjacent non-cancerous tissues showed that the expression of CHAD was significantly reduced in cancer tissues (Fig. 1B). All ten patients were surgical cases from the Department of Breast Surgery at the First Affiliated Hospital of Nanjing Medical University. During the surgery, both cancerous and adjacent non-cancerous tissues were collected from each patient to extract RNA. Postoperative pathology reports indicated that all patients had the luminal subtype. The protein abundance in the triple-negative cell lines was also significantly lower than that in non-triple-negative cell lines, according to the Western Blotting results in 13 samples (Fig. 1C). These results indicate that CHAD is down-regulated in BC tissues, particularly in groups with a high degree of malignancy.

CHAD is a prognostic marker for BC
We also evaluated the prognostic effect of CHAD by dividing the samples into CHAD-high and CHAD-low groups according to the median expression values in the GEO and TCGA-BC datasets. The CHAD-high group had a significantly longer survival time than the CHAD-low group in these two datasets (Fig. 2A and B, respectively). Immunohistochemistry showed that the expression of CHAD protein was lower in triple-negative breast cancer (TNBC) tissues than in luminal-type tissues(Fig. 2C and D). TNBC tends to be more aggressive and proliferative, leading to quicker progression and a higher likelihood of spreading to distant organs. In summary, high CHAD expression is associated with less metastasis and predicts good survival.

Clinicopathological indicators and CHAD
Univariate logistic regression analysis showed that when grouped by the size and depth of invasion of the primary tumor with CHAD expression as the exposure factor, the OR value was less than 1 and the P-value was less than 0.05, indicating that CHAD is a protective factor (Table 1).
Based on the grouping by clinical information, the results of the RT-PCR experiments were referenced. Compared with the tumor recurrence group(N = 28), CHAD expression was significantly higher in the disease-free survival group(N = 32)(Figure3A).Base on the size and depth of invasion of the primary tumor, CHAD expression was the highest in the T1 stage group. CHAD expression was higher in the M0 stage group (Fig. 3B and C). These results indicate that CHAD is an important clinical indicator of prognosis and is associated with cancer differentiation and metastasis.

CHAD opposes proliferation and migration in BC cell lines
Analyses clinical information have revealed that CHAD is associated with metastasis. Overexpressing CHAD in MDA-MB-231 cells weakens cell proliferation capacity. The invasion assay in two cell lines, one is non-triple-negativeT47D and the other is triple-negative MDA-MB-231, was performed. After incubation for 12 h, the number of invading cells was compared (Fig. 4B). The number of invading MDA-MB-231 cells was significantly higher than that of T47D cells. To further substantiate our findings, we examined the migration and proliferation rates of two distinct breast carcinoma (BC) cell lines, T47D and ZR75-30, after CHAD was knocked down using small interfering RNAs (siRNAs) ( Fig. 5A). The proliferation rate was determined with the CCK8 kit every 24 h, demonstrating a marked increase in proliferation capabilities in breast cancer (BC) cells within the CHAD knock-down groups for both lines ( Fig. 5B). We similarly conducted a migration assay post-CHAD knock-down. Following a 12-hour incubation, we compared the number of cells that had migrated (Fig. 5C). In both T47D and ZR75-30 cell lines, there was a notably higher number of migrated cells in the CHAD knock-down groups compared to controls. These findings imply that CHAD’s dysregulated expression is linked to the migration and proliferation of BC cells in vitro.

Pathways associated with CHAD expression
In order to investigate the pathways that CHAD may regulate, GO(Gene Ontology)/ KEGG (Kyoto Encyclopedia of Genes and Genomes) signaling pathways were used as references to evaluate the pathways that CHAD may modulate. For biological processes (BP), CHAD was predominantly enriched in “cell adhesion mediated by integrin” and the “integrin-mediated signaling pathway” (Fig. 6A). Regarding cellular components (CC), CHAD was primarily enriched in the “integrin complex” (Fig. 6B). In terms of molecular function (MF), CHAD was mainly enriched in “integrin binding” and “collagen binding” (Fig. 6C). Carcinogenesis and development associated pathways, including “focal adhesion”, “ECM receptor interaction”, “regulation of actin cytoskeleton”, and “PI3K/Akt signaling pathway” were significantly altered along with aberrant CHAD expression (Fig. 7A). The STRING online tool was used to predict potential interacting proteins with CHAD (Fig. 7B), which showed a strong correlation between CHAD and the ITGA2 and ITGA3 genes. The interaction between CHAD (chondroadherin) and ITGA2/ITGA3 (integrins α2 and α3) can influence several cellular functions, particularly those related to cell adhesion, migration, and signaling. CHAD can bind to integrins, facilitating stronger adhesion to the extracellular matrix, which is crucial in maintaining tissue structure and integrity. By affecting integrin signaling pathways, CHAD can modulate cell movement. Stronger adhesion may reduce cell mobility, while altered interactions might promote migration, relevant in cancer metastasis. Interaction with integrins may activate or inhibit signaling pathways like PI3K/Akt that are involved in cell survival, proliferation, and differentiation. This can impact cancer cell growth and response to the environment. In normal physiology, CHAD-integrin interactions can contribute to tissue development and repair processes, influencing how cells respond to injury. Overall, understanding these interactions provides insight into how CHAD might function as a tumor suppressor and its potential role in cancer progression and therapy. Western blotting was used to detect changes in the expression of proteins related to the PI3K/Akt pathway and cell cycle. The results showed that, compared to the control group, using CHAD siRNA promoted the expression of p-PI3K and p-Akt proteins and promoted the expression of Cyclin D1 (Fig. 7C). In summary, CHAD expression alters the prognosis of breast carcinoma by altering cell adhesion related and PI3K/Akt signaling pathways.

Discussion
The challenge in predicting outcomes and treating breast cancer (BC) lies in the insufficiency of reliable prognostic biomarkers and therapeutic targets. In this study, we analyzed CHAD expression in breast carcinoma (BC) to evaluate its prognostic relevance and examine its impact on cell proliferation and migration. These findings suggest that CHAD could serve as a promising diagnostic and prognostic biomarker for BC because abnormal CHAD expression notably affects the proliferation and migration of BC cell lines. Pathways potentially associated with CHAD expression include “focal adhesion”, “ECM receptor interaction”,“regulation of actin cytoskeleton”and “PI3K/Akt signaling pathway” supporting the notion that CHAD is a viable biomarker for BC prognosis.
CHAD is a cartilage matrix protein that mediates the adhesion of isolated chondrocytes [15–17]. However, there is still limited research on the other functions of CHAD, particularly its role in carcinogenesis and cancer progression. CHAD expression has been detected in both breast carcinoma and normal breast tissues, suggesting that it may influence breast cell behavior. Given that CHAD mediates chondrocyte adhesion, it is suspected that it also participates in cell-cell interactions in breast carcinoma. Functional assessments and clinical correlations revealed that CHAD is downregulated and linked to poor survival and metastasis, which is consistent with this hypothesis. Components such as focal adhesions and ECM receptor interactions are related to breast cancer metastasis. CHAD expression notably altered KEGG signaling pathways, such as those involving focal adhesion, ECM receptor interaction, regulation of the actin cytoskeleton, and the PI3K/Akt pathway, thus supporting our hypothesis.
However, this study is limited by the unclear mechanisms through which CHAD regulates cell-cell interactions. Additionally, the retrospective nature of the study indicates that more extensive research is necessary to validate CHAD’s use of CHAD as a clinical biomarker. In conclusion, our study identified CHAD as a novel prognostic biomarker that is downregulated in breast carcinoma cells, inhibits cell proliferation and migration, predicts favorable survival in BC patients, and by targeting CHAD, it might be possible to exploit its natural tumor-suppressive roles, disrupt cancer-promoting pathways, and enhance the effectiveness of current breast cancer therapies. So it could be a potential therapeutic target.

Electronic supplementary material
Below is the link to the electronic supplementary material.