Recently, APS has attracted growing interests in biological fields especially in terms of cancer therapy. Most of studies have clarified that APS inhibit tumor growth through immunopotentiation activity and generally be considered as a powerful adjunctive strategy in combination with chemotherapy for various tumors [15, 16]. There are also parts of studies suggest the direct anti-tumor activity of APS, such as the inhibition of cell proliferation, the induction of cell apoptosis and cell cycle arrest [17, 18]. In the present study, for the first time, we employed bioinformatics approach to specifically target the common hub genes involved in the in vitro anti-tumor biological processes, and further underwent experimental verification. The aim of this study was to explore other possible anti-tumor mechanisms of APS and to find the potential therapeutic targets for breast cancer.
KEGG pathways analysis demonstrated that the DEGs were mainly enriched in cell cycle, AMPK signaling pathway and pathways in cancer, which are potentially associated in the progression of breast tumor and used as the regulating target for cancer treatment [19, 20]. For instance, downregulation of AMPK activity or decreased expression appear to promote breast tumorigenesis, while AMPK activation was found to impede tumor growth. Pan et al showed that cetyl trimethyl ammonium bromide could enhance DOX chemosensitivity mainly through activation of AMPK signaling cascades in breast cancer . Another report found that targeting AMPK alpha signaling was capable of improving radiosensitivity of triple negative breast cancer . All these potentially enriched pathways will contribute to identify important molecules involved in the growth of breast cancer.
It is more meaningful to study the mechanism of tumor progression by classifying the genes involved in the same function or pathway using GO functional annotation. In this study, PPI networks of all DEGs and corresponding DEGs in four BPs including cell cycle, cell proliferation and cell apoptosis were specially constructed and the hub nodes were selected in all PPI networks. Such treatment focused on the biological process network pathway of differential genes associated with breast cancer rather than being confined to individual genes to study the expression changes of key genes or proteins throughout the entire pathway. Here, the hub nodes were purposefully analyzed in PPI networks constructed by DEGs in 4 typical BPs (cell cycle, cell proliferation, cell apoptosis and death) because they were involved in the anti-cancer mechanism of APS mediated macrophages in our previous study [12, 23]. Moreover, the regulatory genes of cell cycle are frequently linked to the incidence and growth of tumor, which are mutated under the action of carcinogenic factors, causing the disorder of cell cycle followed by the malignant proliferation of cells and tumor formation [24, 25]. It has been extensively acknowledged that cell apoptosis plays a potential role in the inhibition of tumor growth, or it may develop resistance to tumor progression [26, 27]. Further, tumor cell death induced by most of anti-cancer strategies is directly related to the activation of apoptosis. On this basis, the main focus of this study was to investigate the hub genes in the four BP-PPI networks to explore other possible anti-breast cancer mechanisms using bioinformatics based on previous findings in our lab.
In this study, a sum of 56 genes in the four BP-PPI networks, among which 7 genes (CCNB1, DLGAP5, CDC7, CSE1L, GINS1, AURKA and ECT2) were particularly significant with the degree over 45. For these genes, CCNB1 is essential for controlling the cell cycle at the G2/M transition, which has been displayed in multiple cancer types playing important roles in the promotion and transformation of tumor . DLGAP5 and CDC7 are potential cell cycle regulators that play roles in carcinogenesis of cancer cells. Elevated expressions of DLGAP5 were noticed in breast cancer specimens and were associated with poor prognosis . Moreover, a cellular apoptosis susceptibility protein (CSE1L), is extensively expressed in certain types of cancers. Mounting evidences from several pathological reports have been indicated that highly expressed CSE1L are most frequently associated with the tumor proliferation . Tai et al indicated that CSE1L was of paramount importance in facilitating tumor cells apoptosis imparted by chemotherapeutic agents and could also regulate the metastasis and invasion of breast cancer . Accumulating evidence has indicated that the aberrant expression of GINS1 was involved in cancer pathogenesis . Breast cancer patients with elevated expression of GINS1, have been predicted with significantly poor prognosis, however the knockdown of GINS1 could inhibit the malignant features of breast cancer cells . AURKA is involved in regulating many early mitotic events that also plays an important role in tumorigenesis and tumor progression. Extensive studies have demonstrated that AURKA was overexpressed in certain types of malignancies, such as bladder cancer, esophageal squamous cell carcinoma and breast cancer [34, 35]. It is evident from previous studies that ECT2 can improve cell growth, invasion and tumorigenicity . All these findings demonstrate the vital roles of these genes in the progression of breast cancer, and will be verified in our further study.
Previous study in our lab demonstrated that the in vivo the inhibitory activity of APS towards 4T1-solid tumor was partially associated with the enhancement of immune responses . Here, the other in vivo underlying molecular mechanism of APS against breast cancer was further studied using bioinformatics method targeting in vitro anti-tumor strategies. In the present study, common hub genes EGFR and ANXA1 were identified in biological processes involving in cell cycle, cell proliferation and cell apoptosis targeting to the inhibitory mechanism of APS mediated macrophages against breast cancer cells. Correspondingly, IHC and PCR were carried out to study the effect of APS on the proteins and genes expression of EGFR and ANXA1, respectively. Currently, accumulating evidences have demonstrated that EGFR and ANXA1 are aberrant expression in multiple tumors [37, 38]. EGFR, a transmembrane glycoprotein, belongs to the extracellular protein ligand of the epidermal growth factor family that is involved in a myriad of biological processes and carcinogenic events. It can be used as a tumor marker for serological detection. Significant difference of EGFR expression was identified in the serum of healthy people and gastric cancer patients . EGFR can be detected in the serum of patients with early-stage gastric cancer, which thus can be used as one of the screening indicators for high-risk groups. Up-regulated EGFR protein and transcript levels were related to poor prognosis in various cancers, including colorectal cancer, endometrial cancer and non-small cell lung cancer . Besides, EGFR is also a hot target and predictive indicator of early recurrence and death of breast cancer. The degree of EGFRs high expression determines the amount of the down regulation, which occurs in the natural functions of family members (ErbB4/Her4, ErbB3/Her3, ErbB1/EGFR1 and ErbB2/Her2) and their use as indicators for clinical outcome in breast cancer and other tumor types . The study reported that the high levels of surface tyrosine kinase receptors of EGFR family were frequently found in breast cancers . The overexpression of EGFR was identified in approximately 40% of triple-negative breast cancer (TNBC) patients that provide a potential strategy for targeting TNBC therapy . The result of EGFR with high degree indicated its significant role in the progression of breast cancer and reflected the feasibility of PPI analysis.
ANXA1 refers to the first protein found in the Annexins family that plays an extremely vital role in tumor proliferation and differentiation, inflammatory response, cell signal transduction, calcium ion signaling channels, and interactions between cytoskeletal proteins . Currently, numerous reports have demonstrated differential expression of ANXA1 depending on cancer type. Several studies have reported higher levels of ANXA1 in esophageal cancer, skin squamous cell carcinoma and colorectal cancer, while others have reported decreased expression of ANXA1 in head and neck cancer, prostate cancer, oral squamous cell carcinoma and cervical cancer . Significant down-regulation of ANXA1 protein was identified in esophageal squamous cell carcinoma compared with that in paracancerous normal tissue . Notably, there are contradictory reports about the role of ANXA1 plays in breast cancer. Mussunoor et al indicated that the over expression of ANXA1 was found in triple negative breast cancer and metastatic cancer, which plays a prominent role in tumor progression and development , while another report showed that ANXA1 was downregulated during late stage of progression and metastasis in breast cancer . Using high-throughput analysis and also evidenced by IHC and RT-PCR that the event of decreased expression of ANXA1 occurs in the human breast cancer . Besides, previous report showed that the expression of ANXA1 remarkably decreased in breast tumor including ductal carcinoma in situ and invasive carcinoma. Whereas, overexpression of ANXA1 was related to unfavorable prognostic factors in invasive cancer . All these findings suggested that ANXA1 played dualistic roles and was involved in variable mechanisms associated with the advancement and proliferation in breast cancer. In the present study, APS alone especially couple to 5-FU significantly inhibited tumor growth in BALB/c mice after 14 days of treatment. All findings demonstrated that the inhibitory activity of APS towards 4T1-solid tumor probably attributed to the regulation of EGFR and ANXA1 apart from owing to the immunomodulatory role of APS. Most of the previous reports only evaluated the gene and protein expression of EGFR and ANXA1 in vivo in solid-tumor. Here, GFP-4T1 cells were further harvested and analyzed from subcutaneous tumors in all groups. Live/dead staining and CCK-8 analysis manifested remarkable decrease in cell viability after treatment with APS. For the sorted GFP-4T1 cells, it was identifiable that APS down-regulated the expression of EGFR protein and up-regulated the expression of ANXA1 protein using IF and flow cytometry analysis.