HMGA2 Contributes to Distant Metastasis and Poor Prognosis by Promoting Angiogenesis in Oral Squamous Cell Carcinoma

Abstract

The highly malignant phenotype of oral squamous cell carcinoma (OSCC), including the presence of nodal and distant metastasis, reduces patient survival. High-mobility group A protein 2 (HMGA2) is a non-histone chromatin factor that is involved in advanced malignant phenotypes and poor prognosis in several human cancers. However, its biological role in OSCC remains to be elucidated. The purpose of this study was to determine the clinical significance and role of HMGA2 in the malignant potential of OSCC. We first investigated the expression pattern of HMGA2 and its clinical relevance in 110 OSCC specimens using immunohistochemical staining. In addition, we examined the effects HMGA2 on the regulation of vascular endothelial growth factor (VEGF)-A, VEGF-C, and fibroblast growth factor (FGF)-2, which are related to angiogenesis, in vitro. High expression of HMGA2 was significantly correlated with distant metastasis and poor prognosis. Further, HMGA2 depletion in OSCC cells reduced the expression of angiogenesis genes. In OSCC tissues with high HMGA2 expression, angiogenesis genes were increased and a high proportion of blood vessels was observed. These findings suggest that HMGA2 plays a significant role in the regulation of angiogenesis and might be a potential biomarker to predict distant metastasis and prognosis in OSCC.

Keywords: HMGA2; angiogenesis; metastasis; oral squamous cell carcinoma; prognosis.

Figure 1

Tumor high-mobility group A protein 2 (HMGA2) expression status affects the survival rate of oral squamous cell carcinoma (OSCC) patients. (A) Immunohistochemical staining for HMGA2 in OSCC biopsy specimens. Representative microscopic images are shown according to expression status. Original magnification, ×200, scale bar = 100 μm. (B) Based on Kaplan–Meier survival analysis of OSCC, the patients were divided into two groups based on HMGA2 immunostaining scores (low- or high-expression groups). Overall survival (OS) of the 110 OSCC patients based on the status of HMGA2 expression was determined. * p < 0.05. (C) Disease-free survival (DFS) of the 110 OSCC patients based on the status of HMGA2 expression. * p < 0.05.

Figure 1

Tumor high-mobility group A protein 2 (HMGA2) expression status affects the survival rate of oral squamous cell carcinoma (OSCC) patients. (A) Immunohistochemical staining for HMGA2 in OSCC biopsy specimens. Representative microscopic images are shown according to expression status. Original magnification, ×200, scale bar = 100 μm. (B) Based on Kaplan–Meier survival analysis of OSCC, the patients were divided into two groups based on HMGA2 immunostaining scores (low- or high-expression groups). Overall survival (OS) of the 110 OSCC patients based on the status of HMGA2 expression was determined. * p < 0.05. (C) Disease-free survival (DFS) of the 110 OSCC patients based on the status of HMGA2 expression. * p < 0.05.

Figure 2

HMGA2 controls the expression of epithelial–mesenchymal transition (EMT) markers in oral squamous cell carcinoma (OSCC) cell lines. (A,B) The mRNA and protein expression levels of HMGA2 in Ca9-22 cells and SAS cells transfected with HMGA2 siRNA or control. Total RNA and whole-cell lysates were used for qRT-PCR (A) and western blot analysis (B), respectively. (C,D) The mRNA expression levels of E-cadherin, Vimentin, and Slug were measured using qRT-PCR with Ca9-22 cells (C) and SAS cells (D) transfected with si-HMGA2. The values are expressed as the means ± s.d. of triplicate samples. Statistical analysis was performed using a Student’s t-test. * p < 0.05, ** p < 0.01.

Figure 3

HMGA2 regulates the expression of angiogenesis genes in oral squamous cell carcinoma (OSCC) cell lines. (A,B) The mRNA expression levels of VEGF-A, VEGF-C, and FGF-2 were measured using qRT-PCR in Ca9-22 cells (A) and SAS cells (B) transfected with si-HMGA2. The values are expressed as the means ± s.d. of triplicate samples. Statistical analysis was performed using the Student’s t-test. * p < 0.05, ** p < 0.01.

Figure 4

High expression of angiogenesis-associated genes in oral squamous cell carcinoma (OSCC) tissues based on HMGA2 expression. Images of immunohistochemical staining based on the following antibodies in OSCC biopsy specimens: (A) HMGA2, (B) VEGF-A, (C) VEGF-C, and (D) FGF-2. Scale bar, 100 μm.

Figure 5

HMGA2 and angiogenesis-related genes are highly co-expressed at sites of lung metastasis. Images of immunohistochemical staining using the following antibodies in lung metastasis specimens: (A) HMGA2, (B) VEGF-A, (C) VEGF-C, and (D) FGF-2. Representative microscopic images are shown according to the expression status. Scale bar, 100 μm.

Figure 6

Blood vessels are increased in oral squamous cell carcinoma (OSCC) tissues with high HMGA2 expression. The association between blood vessel status, based on C34 staining and HMGA2 expression, as measured by immunohistochemical double staining with OSCC tissues. (A) HMGA2-high expression, (B) HMGA2-negative expression. CD34: green, HMGA2: brown. Scale bar, 100 μm.