Most breast cancer cases are diagnosed at advanced stages or even metastatic stages, leading to a high mortality rate. However, early diagnosis is of great help to the prognosis of patients[26]. Therefore, there is an urgent need to screen out individuals with poor prognoses for targeted treatment. Our previous study has demonstrated that HPSE is a novel biomarker for immune infiltration and prognosis in breast cancer[13]. Therefore, we further analyzed the prognosis value of HPSE and its role in the immune microenvironment of breast cancer. TNM classification, lymph node metastasis, histological type of primary tumor, tumor size, ER status, and tumor biomarkers are recognized as the main factors affecting breast cancer prognosis. According to this study, we further explored the prognostic value of HPSE in patients with breast cancer through the TCGA database. Our results showed that HPSE was associated with OS, PFI, DSS, and DFI, and patients with high expression of HPSE had a poor prognosis. Our study found that the risk of death in patients with high expression of HPSE was 1.69 times higher than in those with low expression. The study by Tang also demonstrated high expression of HPSE in the serum of breast cancer patients, and the expression level of HPSE was utilized as a biomarker to assess the effect and prognosis of the operation[27, 28]. In addition, numerous studies have indicated that HPSE is associated with the poor prognosis of other tumors. For example, in gastric cancer, studies have shown that the prognosis of patients with high expression of HPSE is significantly lower than that of patients with low expression of HPSE. The SNP rs4693608 in HPSE plays a vital role in the survival of gastric cancer [29, 30]. The high expression of HPSE in gastric cancer is a powerful predictor of low survival. In gliomas, tumor development in vivo is positively correlated with HPSE levels, and there is a correlation between high expression of HPSE and shorter survival in patients with highly malignant gliomas[31]. In pancreatic ductal carcinoma, studies have shown a negative correlation between the level of HPSE mRNA in PDAC specimens and postoperative prognostic factors. Patients with high expression of HPSE mRNA have a poor prognosis[32]. In myeloma, HSPE is also associated with shorter event-free survival in newly diagnosed myeloma patients receiving high-dose chemotherapy and stem cell transplantation. It has also been shown that HSPE is associated with shorter event-free survival in newly diagnosed patients with myeloma receiving high-dose chemotherapy and stem cell transplantation.[33]. All of these findings demonstrate that HPSE is closely related to the prognosis of tumor patients. In our model, HPSE can be used as a tumor biomarker for the prognosis of breast cancer patients, and we can predict this by analyzing the expression levels of HPSE and the clinical stage. The results of the ROC curve showed that the 1-year survival rate had an AUC value of 0.747, and the 10-year survival rate was 0.695. The model demonstrated a good predictive effect. The results of the clinical decision curve also indicated that the model provided better clinical benefits. By scoring the patients, we can predict the prognosis and survival rate, enabling clinicians to tailor drug use for different patients and identify the benefits of specific treatment strategies.
Our results also showed that the expression level of HPSE was related to metastasis. We all know that the outcome of metastasis is a poor prognosis. In our study, the expression level of the HPSE gene was high. The possible mechanism was that HPSE cleaves heparan sulfate proteoglycan (HSPG) into endoglycosidase of heparan sulfate side chain and core proteoglycan and then participates in the degradation and remodeling of extracellular matrix (ECM). It becomes active under acidic conditions, promoting cell migration associated with metastasis, wound healing, and inflammation during tumor invasion. In the presence of tissue factors and activating factor VII, it can play the role of a coagulant by increasing the production of activating factor X. HPSE can increase the adhesion of cells to the extracellular matrix (ECM) without affecting its enzymatic activity and induce AKT1/PKB phosphorylation via lipid rafts, increasing cell mobility and invasion[34–38]. Studies also showed that HPSE could induce FAK and ICAM-1-dependent cell adhesion in breast cancer, promoting intravascular cell aggregation. When HPSE is knocked down, or its activity is inhibited by heparanase inhibitor JG6, it can prevent the formation of cell clusters and inhibit breast cancer metastasis[39]. In the breast cancer treatment study, the sequential release of NLC/H (D + F + S) NPs nanoparticles driven by HPSE can regulate the tumor microenvironment, reduce TAF activation and the secretion of TGF-β, and block tumor metastasis[40]. All of these findings further explain that HPSE can regulate breast cancer metastasis and affect the prognosis of patients.
Our study showed that the mutation frequency was higher in Breast Invasive Mixed Mucinous Carcinoma. The expression level of HPSE is related to the expression levels of ER and PR, which means that HPSE may be used as a therapeutic target for triple-negative breast cancer(TNBC) patients, consistent with the study of Yang[41]. HPSE may regulate the metastasis of TNBC through the HIF-1a/VEGF-a/p-AKT axis and then regulate the tumor progression. A study also showed that nanoparticle-HPA-aptamer bioconjugates provided insight into treating TNBC[42], significantly enhancing anti-invasion and anti-angiogenesis activity. In addition, our results showed that HPSE is related to the mutation of the TP53 gene. This may be due to the combination of p53 with the promoter of the HPSE gene[43]. Other studies have shown that p53 mutation with loss of function in tumor cells is associated with the overexpression of HPSE[44]. These pieces of evidence showed that HPSE is a valuable therapeutic target in breast cancer.
Breast cancer treatments include surgery, radiotherapy, chemotherapy, and immunotherapy. Cancer immunotherapy relies on a self-immune response to kill cancer cells and tumor tissues by activating the immune system. HPSE is produced by activated CD4 + T lymphocytes, neutrophils, monocytes and B lymphocytes[45]. In our study, CD8 T cells are highly expressed and strongly correlated with HPSE in breast cancer patients with high HPSE expression. CD8 T cells, also known as cytotoxic T cells (CTLs), are recognized as the most effective anti-tumor immune effector cells[46]. On the surface of T cells, CD8 and MHC-I can specifically recognize tumor-associated antigens and produce perforin and other cytotoxins after binding to tumor cells, killing cancer cells but not affecting normal cells[47]. Therefore, the expression level of HPSE can reflect the expression level of CD8 T cells. In addition, the high expression of HPSE in helper T cells leads to the same high expression of Th1, Th2, and regulatory T cells (Treg). Th1 secretes IL2 and INF- γ, which can activate and promote the proliferation of CD8, T cells, and NK cells[48]. Th2 can promote B cell maturation, cell proliferation and immune cell category conversion thereby changing the humoral immune response (Humoral immune reaction)[49]. The "Th1/Th2" balance pattern and ratio often change in cancer and other diseases. For example, the number of Th2 cells in a cancer patient's blood and tumor tissues is often greater than that of Th1 cells. Therefore, the Th1/Th2 ratio is also often used to predict the survival rate of metastasis and is an important prognostic factor of immune response in the tumor microenvironment[50]. According to our study, the number of Th2 cells was also higher than that of Th1 cells in breast cancer patients with high expression of HPSE. In addition, high expression of HPSE is accompanied by the high expression of B cells, which originate from bone marrow and then migrate to lymphoid organs. After being activated by antigens, they produce antigen-specific plasma cells, which mediate humoral anti-tumor immunity[51]. All of this further confirms that HPSE could accurately predict patient prognosis for breast cancer.
Then we explored the correlation between HPSE and immune checkpoint through in vivo studies. There was a positive correlation between HPSE gene expression levels and immune checkpoint genes as well as immune checkpoint-relevant genes. The physiological function of immune checkpoints is to prevent the attack of autoimmune antigens during immune processes, including PD-1, CD274, or CTLA-4, by reducing immune activation through intracellular signaling mechanisms, This induces T cell degradation and depletion to negatively regulate the recipient immune cells. Tumor cells evade anti-tumor immunity by inactivating cytotoxic T lymphocytes through these pathways. HPSE expression in breast cancer is associated with increased expression of CTLA-4 and CD274 immune checkpoint proteins in our in vivo study. CTLA-4 is expressed in regulatory T cells (Tregs) and up-regulated in activated T cells (ATCs). CTLA-4 is similar to the T cell costimulatory protein CD28 and competes with binding to CD80 (B7), thereby transmitting inhibitory signals negatively and reducing activated CD8 + T cells. This leads to an inability to bind to the corresponding tumor antigen, resulting in immune escape.[52] CD274 is expressed in the tumor cell membrane, antigen-presenting cells (APC), T cells, B cells, etc. After binding with PD-1 in T lymphocytes, CD274 activates downstream inhibitory signals, thereby reducing the activation of CD8 + T cells and inhibiting the immune recognition function. Among these, pro-inflammatory cytokines play a driving role in this link. The increase in pro-inflammatory cytokines in the tumor microenvironment leads to immune regulation of tumor cells, up-regulation of CD274 expression on the cell membrane, and activation of inhibitory signals, allowing tumors to bypass the immune system and eventually progress and metastasize. At the same time, the pro-inflammatory microenvironment attracts Tregs and helps maintain T cell degeneration.[52] Our in vivo experiments show that HPSE positively correlates with the pro-inflammatory cytokine TNF-α in breast cancer. Therefore, the expression level of HPSE can indirectly reflect the level of immune infiltrating cells and immune genes. HPSE can be used as a biomarker of immune infiltration and a prognostic marker of immunotherapy.
We also analyzed the relationship between HPSE and the cancer-immunity cycle, and our results showed that HPSE was highly correlated with seven immune steps. In the cancer-immunity cycle, high expression of HPSE reached 7 immune steps, suggesting that HPSE is closely related to the immune cycle process and participates in the cancer-immunity cycle. According to the mechanism of tumor immune circulation mentioned above, the body can effectively kill tumor cells. However, tumor cells can escape the encirclement and suppression of the immune system through changes of tumor cells themselves or alterations in the tumor microenvironment.
Tumors can express molecules on their surfaces that bind to T cells and inhibit their killing abilities, such as PD1 and CTLA4[53]. Additionally, tumors can secrete cytokines and proteins that inhibit T cells from attacking and killing cancer cells, such as TGF- β, IL-10, and IDO, leading to the loss of T cell activity. Our current immunotherapy includes T cell transplantation, the production of cytokines, regulatory proteins like IL-2, IL-7, and IL-15 that can multiply and strengthen T cells[54]. The other approach involves introducing agonist antibodies, such as OX-40, KANG, anti-GITR, and anti-41BB, as well as neutralization inhibitors such as anti-IL-10, anti-TGF-β, anti-PD1, anti-CTLA4, etc. In this study, the expression of HPSE was up-regulated in tumors, and it was highly correlated with the cancer-immunity cycle and immune cell level. Our results showed that patients with high expression of HPSE are suitable for immunotherapy, and we can also evaluate the status of immunotherapy based on the level of HPSE expression. Additionally, most of the therapy was combined with chemotherapy and immune drugs, so we further predicted the drug sensitivity according to the expression level of HPSE through the website. Among them, 11 drugs included in dasatinib should be noted.
Our results show that HPSE can effectively predict the survival of patients with breast cancer, and it can also evaluate the immune status of patients effectively.. The use of HPSE as a tumor biomarker is reasonable and has the potential to predict clinical outcomes. In other words, HPSE is a valuable biomarker for the treatment and prognosis of patients with breast cancer.