Background
Toxoplasma gondii (
T. gondii) is an obligate intracellular parasite belonging to the phylum
Apicomplexa. Its main forms include oocysts, tachyzoites, cysts and bradyzoites [
1].
T. gondii is predominately divided into three clonal lineages designated type I (highly virulent), II (avirulent) and III (avirulent) [
2,
3]. This parasite has infected approximately one-third of the world's population through digestive tract transmission, blood transmission and congenital transmission [
1,
4‐
6]. Generally,
T. gondii infection is asymptomatic or self-limiting in immunocompetent population, but it can cause severe toxoplasmosis in immunocompromised hosts and severe birth defect in newborns [
7,
8].
However, our recent study found that anti-
T. gondii IgG was associated with a better survival of breast cancer patients, especially in women with high interleukin-17 (IL-17) or IL-9 levels [
9]. Mountains of animal experiments have found that
T. gondii can efficiently inhibit the growth and metastasis of several types of cancer such as ovarian cancer [
10], pancreatic cancer [
11] and breast cancer [
12]. For example, after injecting with artificial attenuated
T. gondii in the mice inoculated with 4T1 cells (murine triple-negative breast cancer cells), tumor growth and metastasis was suppressed by increasing the secretion of interleukin-12 (IL-12) and interferon-γ (IFN- γ), which may inhibit the angiogenesis and induce infiltrating T cells in tumor microenvironment [
12]. In addition, some studies have found that
T. gondii was capable of maturing the dendritic cells, which subsequently activate CD8
+T cells to kill tumor cells [
10].
Recently, a study compared the difference of transcriptome between
T. gondii-infected mice and non-infected ones using RNA-seq. This study found that the expression of several genes related to breast cancer signaling pathway was dysregulated in infected group, such as
BRCA2 (up-regulated) and
CCND1 (down-regulated) [
13].
BRCA2 has been recognized as a tumor suppressor gene [
14], while CCND1 is a possible oncogenic gene. Thus, this study suggests that
T. gondii may reduce the risk of breast cancer by regulating transcriptomic expression. However, it remains unclear how
T. gondii regulates the signaling pathway of breast cancer.
Accordingly, this study assessed the effect of tachyzoites of T. gondii on proliferation and migration of human breast cancer cells using cell growth curve and wound healing assays. Dual RNA-seq was further applied to analyze the transcriptomic changes of breast cancer cells and T. gondii after their interaction.
Discussion
Previous studies using attenuated or wild
T. gondii strain in the treatment of solid tumors, have produced certain therapeutic effects against advanced metastatic cancers, such as ovarian cancer and pancreatic cancer. Recently, our previous study found that anti-
T. gondii IgG could improve the survival of breast cancer patients [
9], but the underlying mechanism was still unclear. In this study, we firstly utilized dual RNA-seq to analyze the transcriptome expression in the interaction between breast cancer cells and
T. gondii.
Our results showed that
T. gondii exerts a time-dependent or a concentration-dependent inhibitory effect on the proliferation of breast cancer cells. These results are consistent with previous studies. For example, Peng et al. found that
T. gondii RH strain not only significantly inhibited the growth of MCF-7 cells after 48 h treating, but also induced the apoptosis of MCF-7 cells [
31]. In our study, Ki-67 levels were decreased significantly in MDA-MB-231 cells infected with RH strain, but not in MCF-7 cells. This result may be due to differences in the ability of the two types of cells to proliferate [
32]. According to a previous research, the expression of Ki-67 in MDA-MB-231 was 100%, while the expression of Ki-67 in MCF-7 cell was only 90% [
32]. Therefore, the expression of Ki-67 might be suppressed more apparently in MDA-MB-231 cells than that in MCF-7 cells. In addition to inhibiting the growth of breast cancer cells,
T. gondii can also significantly inhibit other tumor cells such as prostate cancer cells, esophageal cancer cells, and lung cancer cells [
33]. The underlying mechanisms may be that
T. gondii tachyzoites can induced the G2/M arrest of cancer cells by cyclinB1 [
34].
In addition,
T. gondii can significantly inhibited the migration of breast cancer cells. E-cadherin, as a key protein in cellular adhesion, its down-regulation is linked to cancer progression [
35]. The expression of E-cadherin was significantly different in different breast cancer cell lines [
36]. For example, all breast cancer cell lines except MDA-MB-231 expressed robust levels of E-cadherin [
36]. Because of
T. gondii’ s limited ability to inhibit metastasis, the inhibitory effect on cells with high expression of E-cadherin is limited, while the effect might be apparent in cells which showed low expression of E-cadherin. Moreover,
T. gondii RH strain is a highly virulent strain, while ME49 strain is an avirulent strain, so RH strain might inhibit the migration of breast cancer cells more obviously than ME49 strain. Previous studies have suggested that this inhibiting effect may be closely related to
T. gondii lysate antigens or secreted proteins [
37], such as Toxoplasma profilin (TgPLP) [
38], rhoptry proteins (ROPs) [
39], and dense granule antigens (GRAs) [
39,
40]. For example, TgPLP can increase the level of antigen-presenting cell markers in bone marrow-derived macrophages by activating the MyD88 pathway, resulting in increasing the production of IL-12 and promoting their phagocytosis of tumor cells [
38].
Previous transcriptome sequencing results revealed that
T. gondii significantly altered the expression of transcriptome of host. Both acute and chronic infection with
T. gondii lead to the dysregulation of multiple metabolic pathways [
41,
42], and eventually activated the certain immune response signaling pathways [
43]. In our study, we also found that the transcriptomic expression of breast cancer was significantly changed after interaction with
T. gondii. Particularly, the number of up-regulated and down-regulated genes expressed in MCF-7 cells was significantly higher than that in MDA-MB-231 cells after infection with
T. gondii, which may be due to the heterogeneity of breast cancer cells [
32]. In the present study, KEGG analysis showed the certain signaling pathways were regulated as follow: ribosome, breast cancer pathway, IL-17 signaling pathway, and coronavirus disease (COVID-19) pathway. Particularly, ribosome signaling pathway was also considered as one of the key signaling pathway by GSEA in our study. Ribosome signaling pathway is closely related to cell cycle, especially in G1 phase, a large number of ribosomes are generated, and the generation of ribosomes can promote the progress of cell cycle [
44]. Therefore, when the ribosome signaling pathway is dysregulated by
T. gondii, the cell cycle will be halted, and the normal proliferation of breast cancer cells will be inhibited [
34].In addition, in consistent with the previous studies, our study also found that the breast cancer signaling pathway was significantly dysregulated after interaction with
T. gondii [
13].
Our previous study has found that interleukin-17 (IL-17) was one of the main contributors to the interaction between
T. gondii and breast cancer prognosis [
9]. Likewise, numerous studied have shown that IL-17 plays an important role in promoting tumor proliferation, invasion and metastasis, which is closely related to poor prognosis [
45]. The effects of IL-17 on breast cancer can be divided into direct and indirect effects: IL17 could directly change the gene-expression profile and the behavior of nonmetastatic tumor cells, causing tumor growth in vivo [
46]; IL-17 may also promote tumor progression by recruiting neutrophils to tumor tissue. Neutrophils secrete a variety of proteins that degrade the extracellular matrix, making it easier for tumor cells to invade other sites [
47].
To explore the hub genes in breast cancer cells during the interaction, PPI analysis were further performed.
BRCA1, MYC and
IL-6 were identified as the top three hub genes based on the connectivity. For the gene
BRCA1, it is considered not only playing a vital role on the breast cancer pathway [
48], but also involving in the ribosome biogenesis [
49]. Activation of
MYC [
50] and
IL-6 [
51] has been widely reported in breast cancer progression. However, in our study, how these key genes regulate the signaling pathways still needs further study. Unlike these three top three genes, Early growth response 1 (
EGR1) was further confirmed by qRT-PCR and validated by KM Plotter database in this study.
EGR1 was significantly up-regulated in MCF-7 cells and MDA-MB-231 cells after
T. gondii infection irrespective of strain
. The
EGR1 gene encodes a protein belonging to the early growth response (EGR) protein family, a family of zinc finger transcription factors, which can directly regulate several tumor suppressors such as transforming growth factor beta 1 (
TGFβ1), tumor protein p53 (
p53), and phosphatase and tensin homolog,
PTEN) [
52]. Survival analysis revealed upregulated EGR1 was remarkably associated with favorable relapse-free survival (RFS) among breast cancer patients [
53].
During the interaction of T. gondii and breast cancer cells, not only the transcriptomic expression of breast cancer cells was significantly changed, but also the transcriptome of T. gondii was significantly changed. After dual RNA-seq and annotated by the PHI-base, we found that the expression of ROP16 and ROP18 in T. gondii increased, while the expression of crt, TgIST, GRA15, GRA24 and MIC13 decreased.
ROP16 can polyubiquitinate STING, resulting in inactivate cGAS-STING signaling pathway [
54]. In this way, STING was inactivated and subsequently decreased the secretion of inflammatory cytokines, downregulating the STAT1 and NF-κB pathways in brain metastatic cells, thereby suppressing the brain metastasis of breast cancer and lung cancer [
55,
56].
The secretion of ROP18 and dense granule 24 (GRA24) proteins by
T. gondii activates antitumor immunity through the IL-12/interferon-gamma (IFN-γ) TH1 axis, as well as CD4 + and CD8 + T cells [
39]. However, GRA15 proteins secreted by
T. gondii activated the NF-κB signaling, inducing the secretion of interleukin-6 (IL-6) from immune cells [
57]. IL-6 played pivotal roles in the inflammation, causing the chronic inflammation, which promotes the progression of tumor [
51]. In the present study, we found that the secretion of GRA15 by
T. gondii reduced significantly after interaction with breast cancer cells. Therefore,
T. gondii might suppress the proliferation and migration of breast cancer cells through reducing the secretion of GRA15.
Nevertheless, the present study has some limitations. Firstly, the experiments on the effect of
T. gondii on breast cancer were in vitro experiments, not in vivo experiments. However, the results of the in vitro experiments in this study were consistent with the results of the in vivo experiments in most previous studies [
37]. Further researches would be needed to explore the exact mechanism of
T. gondii suppressing the progression of breast cancer using animal models or human tissue. Secondly, we conducted our experiments only using Luminal A (MCF-7 cells) and triple negative (MDA-MB-231 cells) breast cancer cells, which may not be fully representative of all breast cancer types. Considering the heterogeneity of breast cancer, it is necessary to further explore the association between
T. gondii and breast cancer in multiple breast cancer cells or tissues.
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