High-Mobility Group Box 1 (HMGB1) Promotes Angiogenesis and Tumor Migration by Regulating Hypoxia-Inducible Factor 1 (HIF-1α) Expression via the Phosphatidylinositol 3-Kinase (PI3K)/AKT Signaling Pathway in Breast Cancer Cells

Abstract

BACKGROUND High-mobility group box 1 (HMGB1) is an essential contributor towards initiation and progression of many kinds of cancers. Nevertheless, our understanding of the molecular etiology of HMGB1-modulated vasculogenesis, as well as invasion, of breast cancer is poor. This study explored HMGB1 expression in breast cancer and its role in the development and spread of malignancy. MATERIAL AND METHODS We enrolled 15 patients with breast cancer who received primary surgery at the Department of Thyroid and Breast Surgery in our hospital. HMGB1 was recorded and analyzed. RESULTS Our investigation successfully proves that HMGB1 is upregulated in breast cancer tissues in comparison to the surrounding non-malignant tissues. HMGB1 enhanced vessel formation in breast cancer tissues by regulating hypoxia-inducible factor 1 (HIF-1alpha), which in turn upregulates the expression of VEGF. Furthermore, HMGB1-mediated upregulation of HIF-1alpha relies on its ability to stimulate the phosphatidylinositol 3-kinase (PI3K) pathway to reinforce AKT subunit phosphorylation. HMGB1 overexpression reinforces the vasculogenesis in malignancies not only in vivo but also in vitro. Additionally, shRNA knockdown of HMGB1 prohibited the vessel-forming and invasive capabilities, downregulated VEGF and HIF-1alpha, and suppressed AKT phosphorylation in breast cancer cells. Most importantly, PI3K/AKT axis suppression eliminated the effect of HMGB1-modulated vascularization and invasion in breast cancer cells. CONCLUSIONS Our research indicates that HMGB1 serves as a crucial regulator of malignant cell-modulated vessel formation and is involved in the development of malignancy. Our findings indicate that HMGB1 is a promising target for breast cancer treatment.

Figure 1

HMGB1 is upregulated in BC cells. (A) The qRT-PCR was performed to evaluate the HMGB1 expression in BC tissues as well as surrounding non-malignant tissues (normal). (B–C) Representative immunoblots (B) as well as quantitative assessment of HMGB1 (C) in BC tissues and surrounding non-malignant tissues (normal). Results are stated in the form of mean ±SEM, n=15, ** indicates p<0.01 (compared to normal group).

Figure 2

HMGB1 reinforces the BC cell migration. (A) Migration of MCF-7 cells at the bottom surface of the Transwell membranes in cells transfected with HMGB1 and vector. (B) Quantification of migrated MCF-7 cells in 5 random microscopic fields in different groups. (C) Migratory MCF-7 cells seen at the bottom of Transwell membranes in the cells transfected with negative control (NC) and si-HMGB1. (D) Quantity of migrated MCF-7 cells in 5 random microscopic fields in different groups. Results have been represented in the form of Mean ±SEM, ** p<0.01 (compared to Vector (NC) group).

Figure 3

HMGB1 reinforces malignant vessel generation. MCF-7 cells stably expressing HMGB1, si-HMGB1, or NC were supplemented with Matrigel and injected into both flanks of nude mice. Mice were executed on the 11^th day subsequent to implantation, and Matrigel plugs were trimmed out. (A) Tissues were formalin-fixed and paraffin-embedded, and 5-μm sections were prepared utilizing anti-CD31 antibody; magnification, 400×. Scale bar 20 mm. (B) CD31-positive microvessels were quantified in 3 different fields per section at 400× magnification. Results are shown as mean ±SEM, n=5, * P<0.05 (compared to control group).

Figure 4

HMGB1 enhances/promotes the PI3K/AKT stimulation and HIF/VEGF expression in MCF-7 cells. (A–D) Representative immunoblots (A) as well as quantitative evaluation of AKT phosphorylation (B) HIF (C) and VEGF (D) expression in MCF-7 cells subsequent to transient transfection with HMGB1 plasmid (HMGB1) or vector alone. Results are represented as mean ±SEM. ** P<0.01 (compared to vector group).

Figure 5

HMGB1 KD prohibits PI3K/AKT stimulation and HIF/VEGF expression in MCF-7 cells. (A–D) Representative immunoblots (A) as well as quantitative analysis of AKT phosphorylation (B) HIF (C) and VEGF (D) expression in MCF-7 cells subsequent to transient transfection of HMGB1-specific siRNA (si-HMGB1) or NC. Results are represented as mean ±SEM. ** P<0.01 (compared to NC group).

Figure 6

HMGB1 modulates the malignant vessel formation through PI3K/AKT/mTOR axis in BC tissues. (A) MCF-7 cells stably expressing HMGB1 were supplemented with Matrigel and injected into both the flanks of nude mice prior to treatment with AKT inhibitor (MK2206). Following this, the mice were executed at the 11^th day after injection and the Matrigel plugs were trimmed/excised out. Tissues were then fixed with formalin and embedded in paraffin, followed by sectioning at 5 μm utilizing anti-CD31 antibody; magnification, 400×. Scale bar, 20 mm. (B–D) MCF-7 cells were transfected with HMGB1 prior to treatment with AKT inhibitor (MK2206). Representative immunoblots (B) as well as quantitative evaluation of HIF (C) and VEGF (D) expression in MCF-7 cells. Results are represented as mean ±SEM. ^# P<0.05 (compared to HMGB1 group); ** P<0.01 (compared to control group).