Prox1 and FOXC2 Act as Regulators of Lymphangiogenesis and Angiogenesis in Oral Squamous Cell Carcinoma

et al. (2014) Prox1 and FOXC2 Act as Regulators of Lymphangiogenesis and Angiogenesis in Oral Squamous Cell Carcinoma. PLoS ONE 9(3): e92534. doi:10.1371/journal.pone.0092534 Prox1 and FOXC2 Act as Regulators of Lymphangiogenesis and Angiogenesis in Oral Squamous Cell Carcinoma Tomonori Sasahira 0 Nobuhiro Ueda 0 Kazuhiko Yamamoto 0 Miyako Kurihara 0 Sayako Matsushima 0 Ujjal K. Bhawal 0 Tadaaki Kirita 0 Hiroki Kuniyasu 0 Kaustubh Datta, University of Nebraska Medical Center, United States of America 0 1 Department of Molecular Pathology, Nara Medical University , Kashihara , Japan , 2 Department of Oral and Maxillofacial Surgery, Nara Medical University , Kashihara , Japan , 3 Department of Biochemistry and Molecular Biology, Nihon University School of Dentistry at Matsudo , Matsudo , Japan Prospero homeobox 1 (Prox1) and forkhead box (FOX) C2 regulate angiogenesis and/or lymphangiogenesis. However, the detailed role and function of Prox1 and FOXC2 in cancer remains controversial. In the present study, we examined the expression of Prox1 and FOXC2 proteins in specimens from 163 cases with oral squamous cell carcinoma (OSCC). Furthermore, the role of Prox1 and FOXC2 in cancer cell growth and invasion was evaluated in cultured OSCC cells. Prox1 expression was significantly associated with local progression of the tumor (P = 0.0023), clinical stage (P,0.0001), lymphovessel density (LVD) (P,0.0001), nodal metastasis (P,0.0001), and worse prognosis (P,0.0001). Immunoreactivity of FOXC2 was strongly correlated with microvessel density (MVD) (P,0.0001) and poor prognosis (P = 0.0076). In vitro analysis demonstrated that Prox1 regulates cell growth, proliferation, invasion, and lymphangiogenesis by activating vascular endothelial growth factor (VEGF)-C expression. Furthermore, FOXC2 enhanced the expression level of Prox1 and promoted angiogenesis by enhancement of VEGF-A expression. Our results suggested that Prox1 and FOXC2 play key roles in OSCC progression and that further studies focusing on these proteins may yield useful insights for diagnosis and therapy of OSCC. - Head and neck cancers, including oral squamous cell carcinoma (OSCC), are the sixth most common malignancy in the world [1] and the first leading cause of cancer death in Southern Asia [2]. Every year, 263,900 cases of OSCC and 128,000 OSCC-related deaths are estimated worldwide, [3] and approximately 34,000 patients are diagnosed, representing about 3% of all newly diagnosed cancers in the United States [4]. Moreover, the OSCC mortality rate is 3.7 per 100,000 in Japan [5]. OSCC has a high potential for local invasion and nodal metastasis and over 80% of early stage OSCC patients can be rescued by treatment, whereas less than 70% of advanced stage OSCC patients are incurable. The overall 5-year survival rates of OSCC have not improved significantly in the past 30 years, and it remains less than 50% [6 8]. Therefore, early detection and elucidation of the detailed molecular mechanism of OSCC are important. Prospero homeobox 1 (Prox1) is a mammalian homologue of the Drosophila homeobox protein, prospero [9]. Prox1 is important for the embryonic development of the central nervous system, heart, lymphatic system, skeletal muscles, lens, retina, liver, pancreas, and kidney [10,11]. Prox1 acts as a tumor suppressor in hematologic malignancies [12], esophageal cancer [13], hepatoma [14], pancreatic cancer [15,16], breast cancer [17], and carcinomas of the biliary system [18]. However, recent reports have demonstrated that upregulation of Prox1 is a predictor of poor outcome in colon cancer [11,19], glioma [10], and many vascular endothelial tumors [20,21]. Prox1 is suggested to play various tissue-dependent functional roles, which reflect both an oncogenic potential and a tumor-suppressive role [22]. Thus, the role of Prox1 in malignancies remains controversial. The forkhead box (FOX) transcription factors are a large family of proteins with similar DNA-binding domains [23,24]. Expression of FOXC2 protein was detected in a majority of breast adenocarcinomas, including lobular and ductal adenocarcinomas, and colon adenocarcinoma [25,26]. FOXC2 expression has also been reported in esophageal cancer and could be used as a novel independent prognosis factor [27]. FOXC2 is also an important regulator of epithelial to mesenchymal transition (EMT) in cancer cells [28], while the role of FOXC2 in oral squamous cell carcinoma (OSCC) remains unknown. Angiogenesis and lymphangiogenesis are pivotal for tumor progression and nodal metastasis [29]. The major angiogeneic and lymphangiogenic factors are the vascular endothelial growth factor (VEGF)-A and VEGF receptor (VEGFR) 2 and the VEGF-C/2D and VEGFR3 systems, respectively [30,31]. We previously reported that the VEGF family promotes tumor progression and nodal metastasis by inducing angiogenesis and/or lymphangiogenesis in OSCC [2,29]. More recently, Prox1 was shown to induce lymphangiogenesis by activating VEGFR3 [32]. Prox1 is also a marker for lymphatic endothelial cells [33]. FOXC2 is a regulator of angiogenesis [26] and lymphangiogenesis [34]. Furthermore, Prox1 and FOXC2 are co-expressed and required for the onset of lymphovenous valve formation [35]. In the present study, we examined the expression and role of Prox1 and FOXC2 in human OSCCs. Materials and Methods Surgical Specimens Formalin-fixed, paraffin-embedded 163 cases of primary OSCCs (89 men and 74 women, Age range, 4491 years; means, 66.7 years) were used. We also utilized 15 frozen samples of OSCC (9 men and 6 women, Age range, 5279 years; means, 65.8 64 34 23 0.3713 63 44 14 0.6103 27 60 22 12 0.0023 26 52 27 16 ,0.0001 69 42 14 0.5934 20 59 27 19 0.6915 20 47 34 24 0.8532 9 17 8 4 Relationship between expression of Prox1 or FOXC2 and parameters excluding MVD and LVD were calculated by chi-square test. Relationship between expression of Prox1 or FOXC2 and MVD or LVD were calculated by one-factor ANOVA test. T classification and clinical stage were classified according to the TNM classification. *Histological differentiation: Well, well-differentiated squamous cell carcinoma; Mod, moderately differentiated squamous cell carcinoma; Por, poorly differentiated squamous cell carcinoma. **MVD and LVD were Means 6 S.D. (standard deviation), each S.D. was less than 10% in all cases. doi:10.1371/journal.pone.0092534.t001 Figure 2. Gene expression of Prox1 and FOXC2 by qRT-PCR. The mRNA expression levels of Prox1 (P,0.01) and FOXC2 (P,0.01) in OSCCs were higher than normal oral mucosa. Prox1 expression was upregulated in nodal metastasis positive OSCCs than in those with negative OSCCs (P, 0.01). GAPDH was used for internal control. Error bar, standard deviation (S.D.). doi:10.1371/journal.pone.0092534.g002 years) and 5 cases of non-tumor oral mucosa (3 men and 2 women, Age rage, 3652 years; means, 45.2 years) for gene expression analysis of Prox1 and FOXC2. All specimens were randomly selected from Na (...truncated)