The Clinical Significance Of IL-35 Expression In TILs And Its Biological Effects On Breast Cancer Cells

Background The healthy immune system plays an important role in controlling the progression of malignant diseases. At the same time, immune suppression is the major cause of cancer development and progression. The immune system plays a dual role in cancer development. On the one hand, it could suppress tumor growth by destroying cancer cells or inhibiting their outgrowth; on the other hand, it could promote tumor progression either by selecting tumor cells that are more fit to survive or by establishing conditions that facilitate tumor outgrowth. The recent concept of "cancer immunoediting" defined by three key events:elimination, equilibrium and escape was proposed to elucidate the immune system-tumor interactions. T-regulatory cells (Tregs) are considered to be central mediators of immune suppression and peripheral tolerance. They can promote progression of cancer through immunologic tolerance and ignorance. Interleukin-35 (IL-35) is a novel inhibitory cytokine, which is primarily produced by Foxp3+regulatory T cells (Tregs) and proposed as a key effector molecule of Treg function. In addition to the ability of suppressing effector T cell proliferation and downregulation of Th17 cell development and differentiation, IL-35 also enlarges regulatory responses by generating a potent population of IL-35-producing CD4+Foxp3- induced regulatory T cell population, defined as iTr35 cells. Some diseases have been demonstrated to be associated with increased IL-35 expression, including cancers. Human IL-35 levels in colorectal cancer, pancreatic cancer and NSCLC are associated with the severity and clinical stage.Part 1. The clinical significance of IL-35 expression in TILs and plasmaAim To compare plasma IL-35 levels of patients with breast cancer versus healthy women and further analyze the correlation between IL-35 expression of TILs in breast cancer tissue and patients' clinical characteristics.Methods We examined the expression of IL-35 utilizing immunohistochemistry in 110 newly diagnosed invasive breast cancer patients. Plasma IL-35 also determined in 60 patients with breast invasive ductal carcinoma (IDC) and 30 healthy women by enzyme-linked immunosorbent assay.Results It was shown that 39.1%,43.6% and 17.3% of the 110 patients were absent, weak, and strong IL-35 expression in the TILs. Strong IL-35 expression in TILs was significantly associated with age> 50 years, tumor size> 2 cm, TNM stage ?, and negative ER (All P<0.05). Patients with elevated IL-35 expression in TILs had significantly worse progression-free survival (PFS) and overall survival (OS) than patients with weak or no IL-35 expression (All P<0.05).Patients with IDC had similar plasma IL-35 levels (0.24±0.11 ng/ml) compared with the healthy controls (0.23±0.10ng/ml, P=0.544). Patients with TNM stage ? showed significantly higher plasma IL-35 (0.29 ±0.13ng/ml) than patients with TNM stage ? and ? (0.22±0.09 ng/ml, P= 0.024). In addition, plasma IL-35 levels markedly elevated in patients with positive lymph node metastasis (0.28 ±0.13 ng/ml) compared with patients without lymph node metastasis (0.20±0.05ng/ml, P=0.004).Conclusion Plasma IL-35 level and IL-35 expression in the TILs of breast cancer tissues may be a valuable biomarker in the development and prognosis of IDC.Part 2. Biological effects of IL-35 on breast cancer cells and possible molecular mechanismAim To investigate the biological effects of IL-35 to breast cancer cells including proliferation, apoptosis, cell cycle and related molecules.Methods We utilized MCF-7 cells to investigate the direct effects of IL-35 on breast cancer. MTT assay was used to detect the effects of proliferation. Flow Cytometry was used to detect cell cycles. RT-PCR was used to detect the major proproliferative, anti-proliferative, pro-apoptotic and anti-apoptotic molecules.Results IL-35 promotes proliferation and S-phase cell fraction of MCF-7 cells. IL-35 could alter the expression of the proproliferative, anti-proliferative, pro-apoptotic and anti-apoptotic molecules. IL-35 induced proliferation correlated with an increase in cyclin E and cdk2 and a decrease in p15 expression (P<0.05), while the inhibition of apoptosis was associated with a decrease in TRAIL and Fas (P<0.05). There was no significant difference in other molecules including cyclin B, cyclin D, cdk4, p18, p21, p27, p53, FasL, Bax, FLIP, Bcl-2 and survivin between the experiment and control group (P> 0.05).Conclusion IL-35 could promote growth and inhibited apoptosis in breast cancer cells in vitro by altering the expression of the relevent molecules and be a promising therapeutic target.