Introduction
Study showed that breast cancer is the most frequently diagnosed cancer in 2022 in the USA [
1] and the fifth leading cause of cancer mortality worldwide [
2]. The overall 5-year survival rate for breast cancer patients with metastasis is only 23% [
3]. Breast cancer is highly heterogeneous, and its progression is a complex process that can be influenced by microenvironment and patients’ immune system [
4,
5]. Immune system cells participate in various life activities and exert effects on the clinical outcomes of cancers [
6]. Growing evidence indicated that high level of immune infiltration is correlated with better survival and response to treatment, especially for immunotherapy in breast cancer [
4,
7‐
9].
Coi led-coil domain-containing protein 69 (CCDC69), which locates on 5q33.1, has been demonstrated to play a critical role in controlling the assembly of central spindles and recruitment of midzone component. Recent studies showed that CCDC69 also functions in ovarian cancer [
10], colon cancer [
11], gastric cancer [
12], breast cancer [
13], and lung cancer [
14]. Wang et al. considered CCDC69 as a hub gene related to the immune microenvironment in colon cancer [
11]. Cui et al. revealed that CCDC69 could enhance platinum-induced apoptosis in ovarian cancer [
10], and they further verified that the overexpression of CCDC69 could activate p14ARF/MDM2/p53 pathway and confer cisplatin sensitivity [
15]. Also, CCDC69 has also been reported to be significantly related to the survival of breast patients [
13]. A machine learning study based on TCGA database showed that CCDC69 expression is negatively correlated with tumor purity [
16]. These findings all suggested the prognostic and underlying therapeutic value of CCDC69 in cancers. Currently, comprehensive study of CCDC69 in breast cancer has not been conducted. Moreover, the relationships between CCDC69 and immune infiltration and immunotherapy response in breast cancer remains unclear.
This paper first analyzed the expression and prognostic value of CCDC69 in using clinical breast cancer samples from patients and multiple bioinformatics databases. Protein-protein interaction (PPI) networks were produced. Gene set enrichment analysis (GSEA) was also performed. This study demonstrated the associations of CCDC69 with clinical features, immune infiltration, and immunotherapy in breast cancer. In conclusion, the upregulation of CCDC69 was correlated with favorable prognosis and immunotherapy benefits for breast cancer patients.
Methods and materials
Patients and samples
Breast cancer and adjacent normal tissues were collected after surgery from the First Hospital of China Medical University and it was approved by Ethics Committee of the First Hospital of China Medical University (Number: AF-SOP-07-1.1-01). All the patients were diagnosed clearly by pathologists. Patients diagnosed with other malignant tumors were excluded. We finally collected 36 pairs of tumor and adjacent normal tissues for quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) for differential expression verification. Besides, 101 tumor tissues with follow-up data collected were used for survival analysis.
RNA extraction and qPT-PCR
TRIzol reagent (Invitrogen, USA) was used for the extraction of total RNA. The purity and concentration of the RNA extracts were successively verified by spectrophotometry (A260/A280 ratio should be between 1.8 and 2.0). The Vazyme HIScript II RT SuperMix for qPCR (+ Gdna wiper) Kit was used for the synthesis of cDNA, and Vazyme SYBR Green qPCRmix was used for qPT-PCR. The 2-ΔΔCt method was applied to analyze the relative expression level which was normalized to GAPDH expression. The primers are shown below:
CCDC69 forward: 5′−CTGTCCAGCTCTGTGCATCAGA − 3′,
CCDC69 reverse: 5′−CTGCTCATCCAGTCTGTCTCGA − 3′.
GAPDH forward: 5′−GGAGCGAGATCCCTCCAAAAT − 3′,
GAPDH reverse: 5′−GCTGTTGTCATACTTCTCATGGG − 3′.
Immunohistochemistry
After dehydration and paraffin-embedding, the breast tissues were fixed with 4% paraformaldehyde and prepared as tissue sections. After dewaxing and hydration, we used Citrate buffer for antigen retrieval at 95℃ for 15 min (min). Next, after cooling to room temperature, 3% H2O2 was used to block the endogenous peroxidase activity. Then, the sections were incubated with primary antibody CCDC69 (Novus, NBPI-85,139, 1:200) overnight at 4℃. After that, secondary antibodies incubation, DAB regents (Maxim, DAB-0031/1031) staining, and hematoxylin counterstaining were performed. Two pathologists were invited to evaluate the immunohistochemical results of each section. When disagreement about the results arouse, a third pathologist was invited to independently evaluate the results. After excluding nonspecific staining, cells with clear brown-yellow granulosa in the nucleus or cytoplasm area were defined as positive cells under a microscope.
Assessment of CCDC69 differential expression on clinical samples and bioinformatics platforms
Gene Expression patterns across Normal and Tumor tissues database (GENT2) (
http://gent2.appex.kr/gent2/) is an updated version of GENT providing a user-friendly search of gene expression patterns across different normal and tumor tissues compiled from public gene expression data sets. The current pan-cancer expression analysis was conducted based on GENT2. RNA-seq data of BRCA in level 3 HTSeq-FPKM were downloaded from official TCGA website and further transformed into transcripts per million reads (TPM) format. The expression data based on TCGA database and qPT-PCR outcomes were analyzed by R (version 3.6.3) and R package ggplot2(version 3.3.3) and Graphpad prism(version 8.0.2).
Assessment of the prognosis value of CCDC69 on survival
The Kaplan-Meier Plotter platform (
www.kmplot.com) is an online database including gene expression data and clinical data. With the purpose to assess prognostic value of a specific gene, the platform was applied in drawing the Kaplan-Meier (KM) survival curves for patients with different CCDC69 expression levels [
17]. The R package “survminer (version 0.4.9)” and “survival (version 3.2–10)” was used to analyze patients’ survival data in TCGA database and clinical follow-up dataset of IHC staining group. In the Cox univariate and multivariate regression analysis, factors with a p value more than 0.1 in the univariate analysis were enrolled in the multivariate analysis. R package “survival (version 3.2–10)” was also used in this section.
Identification of differentially expressed genes
R package “DESeq2 (version 1.26.0)” was used to filter differentially expressed genes (DEGs) [
18] (p.adj < 0.05, |log2FoldChange|>2) between high expression group and low expression group of CCDC69 divided by the median value in TCGA database. The R package “ggplot2 (version 3.3.3)” was used to plot the volcano figure.
Protein–protein interaction network
We used STRING (
https://string-db.org) [
19] to examine the interactions (required score (median confidence) > 0.4, FDR stringency (medium) > 5%) among the proteins from DEGs. And we applied Cytoscape and CytoHubba (version 0.1) [
20] to develop a PPI network and identify the top 15 hub genes. GeneMANIA (
https://genemania.org/), which is a flexible user-friendly web site for generating hypotheses about gene function, analyzing gene lists and prioritizing genes for functional assays, was further applied to predict the functions and mechanisms of the selected hub genes [
21].
GSEA of all the detected genes
GSEA software (version 4.0.3) [
22] was used to conduct GSEA for identifying potential enriched functions and pathways of CCDC69-correlated gene set. The c5.all.v7.0.symbols.gmt data sets were downloaded from the MsigDB database (
http://www.broad.mit.edu/gsea/msigdb/index.jsp) on the GSEA website. The default weighted enrichment statistics method was used, and the number of random combinations was set to 1000 times.
Analysis in breast cancer gene-expression miner (bc-GenExMiner) v4.8
The correlations between CCDC69 and ER status, PR status, HER2 status, nodal status, histological types, and PAM50 subtypes were explored using bc-GenExMiner v4.8, which is a statistical mining tool for published breast cancer transcriptomic data [
23].
Immune infiltration analysis
The enrichment score was defined by the single sample GSEA to represent the absolute enrichment degree of a gene set in each sample within a given dataset using R package “GSVA” [
24]. We also calculated the normalized enrichment scores for each immune category. Various immune cell gene set signatures were obtained from a previous study [
25]. We further evaluated the associations between CCDC69 expression and immunomodulators and chemokines in Tumor-Immune System Interactions database (TISIDB) (
http://cis.hku.hk/TISIDB), which is an online integrated repository portal containing abundant human cancer datasets from the TCGA database [
26].
Single cell analysis
We downloaded BC_UNB_10X_E - MTAB – 8107 and TNBC_IMM_10X_GSE169246 breast cancer datasets in h5ad format from IMMUcan database (
https://immucanscdb.vital-it.ch/). And the data were further transferred into rds format by sceasy package. In the follow-up analysis, R package Seurat (version: 4.2.0) was adopted for follow-up analysis. The entire analysis was performed in the R environment.
Immunotherapy response and immune-related score analysis
We detected the expression level of CCDC69 in mouse samples in vivo from immune checkpoint inhibitor (ICI) studies as well as in vitro samples with cytokines treatment from Tumor Immune Syngeneic Mouse database (TISMO) [
27], which is a database for investigating and visualizing gene expression, pathway enrichment, and immune cell infiltration levels in syngeneic mouse models across different immune checkpoint blockade (ICB) treatment and response groups in 23 cancer types. The survival curve and box plots were generated from CAMOIP database, a web server for comprehensive analysis on multi-omics of immunotherapy in pan-cancer (
https://www.camoip.net/).
Drug sensitivity analysis
We explored the predictive value of CCDC69 under different therapeutic strategies for treating breast cancer by Cancer Treatment Response gene signature DataBase (ctr-db) (
http://ctrdb.cloudna.cn/) [
28]. CTR_Microarray_92 and CTR_Microarray_74 were analyzed. The ability to predict drug response was based on the AUC value.
Data presentation and statistical analysis
The quantitative data downloaded from various bioinformatics platforms were shown as the mean plus the standard error of the mean. Shapiro-Wilk normality test, Levene’s test, paired and unpaired samples t test, and Wilcoxon signed rank test were performed to compare the expression between the two groups. For two independent samples, we first used Shapiro-Wilk normality test and Levene’s test to assess the normality and homogeneity of variance, and if they all met the criteria, unpaired t test was applied, otherwise Wilcoxon signed rank test was used. For paired samples, Shapiro-Wilk normality test was first used to test the normality, and if the samples were normally distributed, paired t test was used, otherwise Wilcoxon signed rank test was used. Spearman correlation test was performed to evaluate the correlations in the immune infiltration analysis. In the survival analysis, univariate and multivariate Cox regression models were employed to investigate the relationship between clinical factors and survival. Survival curves were compared by log-rank test. And the p < 0.05 was considered as statistically significant. And Graphpad Prism 8.0.2 was used to visualize the qPT-PCR results and the data downloaded from TISMO database. All the other statistical analyses were performed using R software (version 3.6.3).
Discussion
Breast cancer is clinically divided into four molecular subtypes, namely, luminal A and B; HER2-positive, and triple-negative breast cancer (TNBC) by the expression of ER, PR, HER2, and KI-67, but such a classification cannot fully realize personalized precision medicine for treating breast cancer. More targets and biomarkers and more precise molecular subtyping should be explored to improve therapeutic efficacy and reduce adverse side effects. With the continuous development of sequencing platforms, in-depth bioinformatics analysis based on genomic data has been increasingly applied for biomarker prediction, prognosis analysis, and targeted therapy in cancers as well as some other diseases [
29‐
32]. In this study, we conducted a series of bioinformatics analyses on the basis of multiple bioinformatics databases and further verified the results in clinical samples. We found that CCDC69 was a downregulated gene in breast cancer tissues compared with normal tissues, and demonstrated the prognosis value of CCDC69 and its protective effects on breast cancer from multiple aspects. CCDC69 is expected to be an effective biomarker to predict the survival of breast cancer patients, facilitating the early diagnosis based on molecular subtypes, histological subtypes as well as lymph nodes metastasis of breast cancer. Besides, the expression of CCDC69 is also a useful predictor of immunotherapy response in multiple cancers. Therefore, personalize treatment and management strategies can be developed appropriately based on the combination of CCDC69 expression level and other factors.
It is known that tumor immune infiltration could affect the sensitivity to chemotherapy, radiotherapy, immunotherapy and also the survival of cancer patients [
33‐
35]. In our research, we detected strong correlations between CCDC69 expression and multiple immune cells infiltration. The favorable effects of T cells including CD8 + T cells [
36,
37] and some subtypes of CD4 + T cells such as TFH [
38] and Th1 [
38] in breast cancer have been revealed. DCs act as a tumor antigen transporter to initiate T cell activation, which is required for T cell-dependent immunity and response to ICI therapy [
39,
40]. Moreover, the anti-tumor effects of B cells [
41,
42], eosinophils [
43], and NK CD56dim cells [
44] in breast cancer have been proven. However, the biological functions of neutrophils [
45,
46] and mast cells [
47] in breast cancer are still controversial. The specific roles of CCDC69 in the biological processes of neutrophils and mast cells in breast cancer are still under exploration, and future study could analyze the function of the both cells in breast cancer. These results revealed that high expression of CCDC69 indicated favorable prognosis in breast cancer possibly through promoting T cells proliferation and activation and anti-tumor immunity.
Due to the emerging role of immune system in breast cancer progression and prognosis, immunotherapy, especially ICIs, has become a hot research subject [
48]. The antibodies of programmed cell death receptor 1 (PD-1), programmed cell death 1 ligand 1 (PD-L1), and cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) have been applied as ICIs for the treatment of breast cancer. The immunotherapy response in breast cancer is associated with T cell infiltration [
48], while higher T cell infiltration level predicts better ICIs treatment response [
49]. In our results, higher CCDC69 expression suggested more ICIs treatment benefits, and we speculated that CCDC69 can improve immunotherapy efficiency by promoting the activation of T cells. Moreover, IFN, a kind of cytokine, has been applied in the immunotherapy of cancers [
50]. In breast cancer, the major source of IFN-gamma is Th1 cells and CD8 + T cells [
51]. The production of IFN-gamma can boost anti-tumoral T cell response [
52,
53]. We observed that the application of IFN-gamma upregulated the expression of CCDC69 in vitro in our results, and CCDC69 possibly participated in the regulatory process when IFN-gamma activating T cell responses.
Currently, ICIs targeting PD-L1 has been widely used as an effective therapeutic option for treating TNBC patients [
54]. However, the clinical practice of ICIs in the therapy of ER/PR + breast cancer patients was not satisfactory [
55]. Our results indicated that the CCDC69 was downregulated in ER/PR + breast cancer samples, while the upregulation of CCDC69 was correlated with high level of TILs, especially T cells, in breast cancer. Accumulating evidence has shown a favorable value of TILs in the prognosis of TNBC and HER + breast cancer patients, but the role of TILs in luminal breast cancer was still unclear [
56]. A deeper understanding of CCDC69 and its effects on the regulating of immune infiltration could help improve the therapeutic effect of ICIs on luminal breast cancer.
In a word, CCDC69 was downregulated in breast cancer, and it was correlated with a better clinical prognosis. Our results demonstrated that CCDC69 regulated multiple immunity-related mechanisms and affected the immune cell infiltration, especially T cells and DC cells, in breast cancer. Moreover, CCDC69 played important roles in the immunotherapy responses and higher expression level predicted better immunotherapy responses. Further researches could be conducted to explore the exact mechanisms of CCDC69 in breast cancer immune microenvironment regulation and immunotherapy response.
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