Unveiling the Role of SYNGR4 in Breast Cancer Development: A Novel Target for Immunotherapy

doi:10.21203/rs.3.rs-4177295/v1

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Unveiling the Role of SYNGR4 in Breast Cancer Development: A Novel Target for Immunotherapy

https://doi.org/10.21203/rs.3.rs-4177295/v1

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Although SYNGR4 is recognized as a contributing gene to amyotrophic lateral sclerosis, its involvement in the progression of breast cancer remains unknown. SYNGR4 expression in multiple types of cancer, such as breast cancer. The effect of SYNGR4 on breast cancer prognosis was analyzed using bioinformatics and possible pathways by which this molecule affects breast cancer prognosis were explored. GSVA was used to analyze the impact of SYNGR4 on immune infiltration in breast cancer, while cellular and animal experiments confirmed the influence of SYNGR4 on breast cancer proliferation, migration, and tumor-associated macrophage polarization within cancer foci. SYNGR4 is abundantly present in numerous cancerous growths, such as breast cancer, and has an impact on the outlook for individuals with breast cancer. This may be a volatile effect through Organelle fission, chromosome segregation, nuclear division, etc. Increased SYNGR4 expression impacts the growth, movement, and immune cell infiltration of breast cancer, as well as encourages the transformation of tumor-associated macrophages in breast cancer towards M2 polarization. SYNGR4 overexpression can affect the prognosis of breast cancer patients by promoting M2 polarization of tumor-associated macrophages in breast cancer, and this molecule may be a novel target for breast cancer immunotherapy.

Health sciences/Biomarkers/Prognostic markers

Biological sciences/Cancer/Breast cancer

Biological sciences/Cancer/Oncogenes

Biological sciences/Cancer/Tumour biomarkers

Biological sciences/Cancer/Tumour immunology

breast cancer

SYNGR4

Cell proliferation

Immune infiltration

Oncogene

Breast cancer has become the most common cancer globally, surpassing lung cancer in recent years and posing a growing threat to the well-being of a larger population. Conventional methods for treating breast cancer involve surgical procedures, radiation therapy, chemotherapy, hormone therapy, and targeted treatments. Early detection and consistent diagnosis and treatment have significantly enhanced the outlook for individuals with breast cancer. However, the effectiveness of breast cancer therapy remains a major area for enhancement due to tumor diversity and resistance to medication. (5)

Immunotherapy is considered the most promising treatment for eliminating cancer, with numerous ongoing studies and clinical trials focused on breast cancer immunotherapy. Despite this, the limited effectiveness of current treatments has hindered the widespread adoption of breast cancer immunotherapy. The identification of novel immunotherapy targets is crucial in the age of precision medicine to enhance the outlook for individuals with breast cancer and reduce healthcare expenses. (9, 10)

SYNGR4 is a member of the SYNGR protein family, with SYNGR1 being predominantly found in neurons of the central nervous system and to a lesser extent in other tissues. It is believed to be linked to schizophrenia and bi-directional affective disorders. SYNGR2 is highly expressed in all tissues except the brain, and is believed to function as an oncogene in esophageal squamous cell carcinoma and hepatocellular carcinoma. SYNGR3 shows high levels of expression in the brain and is significantly upregulated in Parkinson's disease and Alzheimer's disease, with some evidence suggesting it may also be a contributing factor in Head and Neck Cancer. (15-18)

SYNGR4 is believed to be the gene responsible for amyotrophic lateral sclerosis, but its involvement in breast cancer remains unclear.

Clinical samples from patients

Jingzhou Central Hospital collected 10 paired cases of breast cancer tissues and paracarcinoma tissues from June 2023 to December 2023.The samples were stored in a negative 80 degree Celsius environment. Written informed consent was obtained from all participants after approval by the Ethics Committee at Jingzhou Central Hospital. All procedures were conducted in accordance with the ethical principles of the “Declaration of Helsinki”. Informed consent by the study participant or a legally authorized representative was given prior to inclusion in the study.

Survival analysis in the TCGA database

Based on the levels of SYNGR4 expression, breast cancer samples from the TCGA dataset were divided into groups with high and low expression. Kaplan-Meier plots were used to analyze the prognostic variances of these two groups. Functional enrichment analysis

We identified 1290 genes correlated with SYNG4 co-expression in BC using the clusterProfiler package in the R software (|FoldChange| > 1.5 and P ＜0.05). On the basis of the 1482 genes associated with the prognosis of BC, GO/KEGG pathway analysis was performed on the 104 genes, followed by visualization using Graphpad Prism.

Cell Culture and Treatment：

The cell lines SKBR3, MCF-7, MDA-MB-231, and MCF-10A were acquired from the Wuhan Institute of Cell Biology in Wuhan, China. A ThermoFisher incubator was maintained at 37°C with 5% CO2.When STR analysis is performed on a database, every acquired cell line can be identified.

Pathological Sample Processing：

Tumors and surrounding tissues were exposed to a 10% formalin solution, then encased in paraffin, sliced into 5 mm sections, and underwent dewaxing, rehydration, and microwaving. Following a 30-minute incubation at 1°C, the samples were treated with SYNGR4 antibody from Thermofisher Scientific (PA5-20879) at room temperature. Subsequently, the secondary antibodies were labeled with DAB substrate and hematoxylin.

RNA Extraction and qRT-PCR：

Total RNA was isolated using TRIzol reagent with SYNGR4 and GAPDH primers purchased from Tsingke Biotech. The primer sequences for SYNGR4 were as follows: forward ACCGACGGCTACCAGAACA and reverse GAGCTGGAAGGCTGTCTTGA.PCR was performed 40 times at 95°C for 5 minutes, then normalized to the internal control and analyzed using a 2-ΔΔCT approach.

CCK8 Assay：

was determined using an enzyme marker at 450 nm. After digesting the cells, they were resuspended in full culture media, and cell growth was determined Optical density using the CCK-8 assay at 24, 48, 72, and 96 hours.

Colony-Formation Assay：

6-well plates were inoculated with a total of 5,000 cells. Following fixation in 4% polyacetal for 10 minutes, colonies were dyed with crystal violet. Photographs and tallies of colonies were taken.

Transwell Assay：

Using a transwell chamber from Corning in the USA, 20,000 cells were removed from the upper surface after incubating for 24 hours at 37°C. Subsequently, the cells were stained with crystal violet for 10 minutes, followed by counting and imaging of the migrating cells.

scratch test:

IBIDI dual-well culture inserts were placed in 24-well plates with SKBR3, MCF-7, and MDA-MB-231 breast cancer cells and incubated for 24 hours. The inserts were carefully extracted from the pristine surface using forceps, then 1mL of low serum media was added to each well. The wells were then observed under a light microscope on days 0 and 1 following insert removal.

Tumor Xenograft：

Each group consists of twelve female nude mice without thymus, aged 8 weeks with an average weight of 25 grams. A total of one thousand cells were subcutaneously implanted into the lateral abdomen, each group containing 12 mice. On every other day, the xenografts were measured to determine their ultimate size. The ultimate size was calculated as V = 0.5 × L (tumor length) × W² (tumor width). We observed every nude mouse for 20 days before giving them anesthesia until they died, then removing their tumors and weighing them. All procedures were conducted in compliance with the guidelines approved by the Animal Care Committee of Jingzhou Central Hospital (Approval Numbers: JZ2023199) and were performed in accordance with ARRIVE guidelines.

Immune Cell Infiltration：

Immune cell infiltration in BC was analyzed with the GSVA package in R, and the results were generated with the ssGSEA package. In TCGA-BRCA samples corresponding to high or low immunity cells infiltration, SYNGR4 expression was determined by categorizing 24 immune cells and consulting previous research.

Immunofluorescence microscopy：

DAPI staining and paraformaldehyde exposure for 15 minutes enabled the identification of actin and nuclei in cells. After staining the cells, they were examined under fluorescence microscopy.

ELISA：

In a coculture of BC cells and macrophages, levels of specific cytokines were measured by ELISA in the culture medium obtained from the lower chamber. We calculated the 450 nm absorbance using an enzyme marker and expressed it in picograms per milliliter using a standard curve.

Human and animal rights:

The protocol of animal and human samples study was approved by Ethics Committee of Jingzhou Central Hospital affiliated to Yangtze University (approval no. JZ2023199).

SYNGR4 expression analysis

SYNGR4 was found to be upregulated in breast cancer samples, both unpaired and paired, based on data from the TCGA and GTEx pancancer databases. In the pan-cancer analysis, SYNGR4 was overexpressed in 12 malignant tumors including breast cancer. (Figure 1C) This result was verified by the paired samples of breast cancer and paracancer collected at our center. Typical immunohistochemical pictures and quantitative analysis are shown in Figure2A-B.

Breast cancer patients' prognosis and SYNGR4 expression

We examined how SYNGR4 impacts the outlook for breast cancer by studying its correlation with OS (overall survival) (HR=1.59, P=0.004) (Figure 3A), DSS (disease specific survival) (HR=1.74, P=0.011) (Figure 3B), and PFI (progress free interval) (HR=1.64, P=0.003) (Figure 3C) in patients with breast cancer. Correlations were observed between OS (overall survival) (HR=1.64, P=0.003) and PFI (progression-free interval) (Figure 3C) (Figure 3B). Figure 3D shows the ROC curve for SYNGR4-related breast cancer diagnosis.(AUC=0.882)

Correlation and enrichment analyses

SYNGR4 co-expressed genes were identified in the TCGA-BRCA dataset, and the top 25 genes showing positive and negative correlations were visualized in heat maps labeled as Figure 4A and Figure 4B, correspondingly. Gene sets with absolute values greater than 1.5 and P values less than 0.05 were chosen as co-expressed genes for GO/KEGG analysis, with the findings presented in Figure 4C.co-expressed genes and molecular genes associated with breast cancer prognosis-related molecular gene sets were taken to intersect (Figure 4D), 104 genes were obtained, and then GO/KEGG analysis was performed (Figure 5A), and the results suggested that the possible pathways by which SYNGR4 affects the prognosis of breast cancer patients are Organelle fission, chromosome segregation, nuclear division, etc. These 104 genes were selected for protein interactions analysis, and 15 molecules were screened by selecting the highest confidence level to visualize their protein interactions network (Figure 5B) and correlation heatmap (Figure 5C). This network may be a collection of molecular pathways of SYNGR4 affecting breast cancer prognosis.

Expression of SYNGR4 and immune cell infiltration

Patients from the TCGA-BRCA dataset were split into two categories based on median SYNGR4 levels for studying immune cell infiltration. Figure 6A displays the infiltration of 24 immune cells. With SYNGR4 overexpression, Th2 cell infiltration increased (Figure 6B), while Tcm (Figure 6C), macrophages (Figure 6D), and CD8-positive T cell infiltration decreased. (Figure 6E) Based on the TIDE algorithm to predict the responsiveness of breast cancers with different SYNGR4 expression groups to immunotherapy, breast cancers with SYNGR4 overexpression had a higher responsiveness to immunotherapy. (Figure 7)

Knocking down SYNGR4 inhibited malignant behavior in breast cancer cells

SYNGR4 showed significantly higher levels of expression in SKBRE3, MCF-7, and MDA-MB-231 cell lines compared to the MCF-10A cell line, which serves as a model for normal breast cells (Figure 8A). Knockdown of SYNGR4 in three breast cancer cell lines using two si-RNAs yielded satisfactory knockdown efficiencies. SYNGR4 knockdown decreased the malignant characteristics of breast cancer cells, including proliferation and migration, as shown in the clone formation assay (Figure 8B-D). Colony-formation assay (Figure 9A), Transwell assay (Figure 9B), and scratch assay (Figure 9C). Multiple repetitions of the aforementioned experiments were conducted and subsequently subjected to statistical analysis (Figure 9D-F). CCK8 assay shows that knockdown of SYNGR4 significantly inhibits the proliferative capacity of various breast cancer cell lines (Figure 9G-I). In vivo experiments showed that SYNGR4 knockdown breast cancer had reduced tumorigenicity in vivo. (Figure 9J-K)

SYNGR4 knockdown promotes tumor-associated macrophage M1 polarization in breast cancer

To mimic the environment of tumor immune microenvironment in which breast cancer cells interact with tumor-associated macrophages, we constructed a co-culture model as shown in Figure 10A.After 36 hours of co-culture, the lower chamber's cell culture fluid was gathered for cytokine level assessment. Compared to the control group, pro-inflammatory cytokines like IL-1β, IL-6, and TNF-α increased, while anti-inflammatory cytokines IL-4 and IL-10 decreased in the SYNGR4 knockdown group. (Figure 10B) Macrophages were collected and assayed for the expression of polarization markers in them. iNOS, a marker of M1 activation, along with TNF-α and IL-1β, showed increased levels in the group with SYNGR4 knockdown, whereas Arg-1, IL-10, and TGF-β, markers of M2 activation, exhibited decreased levels. Immunofluorescence maps showed that macrophages in the SYNGR4 knockdown group were in the M1-polarized morphology, while the control group tended to be in the M2-polarized morphology.

The frequency of breast cancer has significantly increased in the last forty years. In 2040, over 3 million new instances of breast cancer are anticipated annually, resulting in more than 1 million fatalities each year. (22) After decades of continuous research, researchers have developed a variety of new therapies in addition to traditional treatments for breast cancer. PARP inhibitors like Olaparib, CDK4/6 inhibitors such as palbociclib, abemaciclib, and ribociclib have received FDA approval for treating breast cancer. Clinical trials are currently testing AKT inhibitors like MK-2206 and angiogenesis inhibitors like bevacizumab. However, the optimal treatment for all subtypes of breast cancer has not yet been identified. Hence, identifying novel genes responsible for breast cancer and finding new targets for treatment are crucial for effectively treating breast cancer.

With advances in immunology, there is a growing understanding of tumor escape immune cell surveillance and killing. Recent recognition of immunotherapy as a potential treatment for breast cancer involves targeting specific proteins expressed in cancer cells, showing promise for therapy. Immune checkpoints can influence T cell activation and tolerance. Inhibitory signals from the PD-1/PD-L1 axis and CTLA-4 can hinder T-cell immune responses, preventing the elimination of tumor cells and potentially promoting tumor growth. Drugs like Pembrolizumab, Nivolumab, Avelumab, Atezumab, and Durvalumab have been created for breast cancer immunotherapy, but their effectiveness is restricted by PD-1/PD-L1 expression, limiting the number of patients who can benefit from them.

Increasing understanding of the tumor immune microenvironment has led to the realization that the interaction between tumors and immune cells within cancer foci may offer a new approach for treating cancer. Our study revealed that elevated levels of SYNGR4 led to enhanced infiltration of Th2 cells in the immune microenvironment of breast cancer. Both Th1 and Th2 cells originated from the precursor cell Th0, with increased Th2 cells capable of producing various cytokines like IL-4 and IL-10. This created an anti-inflammatory environment that facilitated tumor evasion from immune destruction. Meanwhile, the reduction of Tcm (Central Memory T cell) and macrophages, CD8-positive T cells can reduce the long-term and short- and medium-term tumor killing ability of immune cells, respectively. Thus, SYNGR4 has the potential to reverse this Th2/Th1 drift and T cell depletion. In this study, we found that reducing SYNGR4 expression leads to a shift in tumor-associated macrophages towards the M1 (pro-inflammatory) phenotype in breast cancer, potentially aiding in the reversal of the anti-inflammatory tumor immune microenvironment. Macrophages, originating from monocytes, are crucial for both innate and adaptive immunity.

Thus, SYNGR4 could serve as a promising target for immunotherapy in breast cancer and warrants additional research.

The shortcoming of the non-study is the discrepancy between the co-culture model and the real, complex tumor immune microenvironment, and further in vivo experiments are needed to validate the therapeutic potential of SYNGR4.

Acknowledgments

Not applicable.

Ethical Approval

The protocol was approved by the Ethics Committee of Jingzhou Central Hospital affiliated to Yangtze University. (No. JZ2023199), and all procedures were performed in accordance with the ethical principles of the “Declaration of Helsinki” and ARRIVE guidelines (https://arriveguidelines.org).

Patient consent for publication

The participant gave written informed consent before the collection of specimens. All patients gave consent for the article to be published.

Authors' contributions

JM and YY obtained clinical specimens; JM performed the data analysis; YY performed the formal analysis; ZG and HW performed immunohistochemical analysis; HW and JM wrote the manuscript. All authors reviewed the manuscript.

Competing interests

The authors declare no potential competing interest.

data availability statement

The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.

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No competing interests reported.

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Unveiling the Role of SYNGR4 in Breast Cancer Development: A Novel Target for Immunotherapy

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