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

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.


Introduction
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][7][8]. Therefore, early detection and elucidation of the detailed molecular mechanism of OSCC are important.
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.

Surgical Specimens
Formalin-fixed, paraffin-embedded 163 cases of primary OSCCs (89 men and 74 women, Age range, 44-91 years; means, 66.7 years) were used. We also utilized 15 frozen samples of OSCC (9 men and 6 women, Age range, 52-79 years; means, 65.8 Figure 1. Immunohistochemical analysis of Prox1 and FOXC2 in human OSCC cases. Prox1 (a) and FOXC2 (b) expression were not observed in non-tumor oral mucosa. The lymphoendothelial cells (arrow) showed immunoreactivity to Prox1 (c) and expression of FOXC2 was found in lymphoendothelial cells (arrow) and vascular endothelial cells (arrow head) (d). Expression of Prox1 (e) and FOXC2 (f) were observed in cytoplasm of the cancer cells. LYVE1 positive lymphovessels (g) and CD34 positive blood vessels were counted for MVD and LVD, respectively. Original magnification was 200-fold. Bar years) and 5 cases of non-tumor oral mucosa (3 men and 2 women, Age rage, 36-52 years; means, 45.2 years) for gene expression analysis of Prox1 and FOXC2. All specimens were randomly selected from Nara Medical University Hospital, Kashihara, Japan. All cases were performed without pre-operative therapy. Tumor staging and the histological grade of OSCCs were classified in order to UICC TNM classification system, 7th edition and WHO criteria, respectively.

Ethics Statement
This study was approved by the Medical Ethical Committee of the Nara Medical University (approval number. 719). Medical records and prognostic follow-up data were obtained from the patient database maintained by the hospital. The study protocol using human samples were performed according to the ethical standards expressed in the Declaration of Helsinki. Written informed consent was obtained from individual patients for use of their tissue samples. For strict privacy protection, identifying information for all samples was removed before analysis.

Immunohistochemistry
Consecutive 3 mm sections were cut from each block, and immunohistochemistry was performed as we described previously. An immunoperoxidase technique was done following antigen retrieval with microwave treatment (95uC) in citrate buffer (pH 6.0) for 45 min. After endogenous peroxidase block by 3% H 2 O 2 -methanol for 15 min, specimens were rinsed with phosphate-buffered saline (PBS) three times. Anti-Prox1 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-FOXC2 antibody (Santa Cruz Biotechnology), anti-CD34 anti-body (a marker for vascular endothelial cells) (DAKO, Carpinteria, CA, USA), and anti-LYVE-1 antibody (a marker for lymphovascular endothelial cells) (Abcam, Tokyo, Japan) diluted by 0.5 mg/ml were used for primary antibody. After 2 h incubated at room temperature, specimens were rinsed with PBS three times and treated for an hour at room temperature with the secondary antibody peroxidase-conjugated anti-mouse (Medical & Biological Laboratories Co., Ltd., Nagoya, Japan) diluted at 0.5%. The specimens were then rinsed with PBS three times and colordeveloped with diaminobenzidine (DAB) solution (DAKO). After washing, specimens were counterstained with Meyer's-hematoxylin (Sigma Chemical Co., St. Louis, MO, USA). Immunostaining of all samples was performed at the same conditions of antibody reaction and DAB exposure.

Evaluation of Immunohistochemistry
Immunoreactivity of Prox1 and FOXC2 were classified according to Allred's score (AS) [36] and we divided the immunoreactivity into 4 grades by AS; Grade 0, AS is 0; Grade 1, AS is 2 , 4; Grade 2, AS is 5, 6; Grade 3, AS is 7, 8. Cases with Grade 2 and 3 were regarded as immunologically positive [2]. The microvessel density (MVD) and lymphovessel density (LVD) were measured on anti-CD34 and anti-LYVE-1 antibody immunopositive specimens, respectively. To quantify MVD or LVD, 5 maximum vessel density fields were selected from around of the tumor cells (the 'hot spot') and examined under a 200-fold magnification by microscope and averaged. We divided the tissue samples into two groups according to MVD levels; those with values higher than the mean value for the entire group, and those

Quantitative Reverse Transcription-polymerase Chain Reaction
Total RNA was extracted using RNeasy Mini Kit (Qiagen Inc., Valencia, CA, USA) and total RNA (1 mg) was synthesized with the ReverTra Ace qRT Kit (Toyobo, Osaka, Japan). Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) were performed on StepOne Plus Real-Time PCR Systems (Applied Biosystems, Foster City, CA, USA) using TaqMan Fast Universal PCR Master Mix (Applied Biosystems) and analyze the relative standard curve quantification method. PCR condition was according to the manufacturer's instructions and GAPDH mRNA level was amplified for internal control. TaqMan Gene Expression Assays of Prox1, FOXC2, VEGF-A, VEGF-C, VEGF-D, and GAPDH were purchased from Applied Biosystems. All PCRs were done at triplicate.

Cell Growth Assay
The cells were seeded at density of 2,000 cells per well of 96-well tissue culture plates and incubated for 48 h at 37uC. Cell growth was assessed by MTT assay using the incorporation of 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma Chemical Co.). The experiments were performed in triplicate.

In vitro Invasion Assay
A modified Boyden chamber assay was done using the BD BioCoat Cell Culture Inserts glued to type IV collagen (Becton-Dickinson Labware, Bedford, MA, USA) as described previously. Briefly, cells were suspended in 500 ml of DMEM and placed in the insert. After 48 h incubation at 37uC, the filters were stained with hematoxylin. The stained cells were counted in whole inserts at 1006magnification. Each experiment was repeated at least three times.

Cell Growth of Endothelial Cells Treated with Conditioned Medium from OSCC Cells
To generate conditioned media, 1610 5 negative siRNA, Prox1 siRNA, or FOXC2 siRNA treated KON cells were seeded in 24well plates. After 24 h incubated at 37uC, the culture media were collected and centrifuged at 1,500 rpm for 5 min to remove pellet and collected the supernatants. HUVECs and HDLMVECs were seeded at a density of 2,000 cells in 96-well plates and incubated to overnight. Cells were then cultured with only Endothelial growth media, Microvacular endothelial growth media, or conditioned media (negative siRNA, Prox1 siRNA, or FOXC2 siRNA treatetd KON media and Endothelial growth media or Microvacular endothelial growth media (1:1)). After 48 h, growth ability of endothelial cells was measured with the MTT assay [37].

Migration of Endothelial Cells Co-cultured with OSCC Cells
The in vitro endothelial cell migration assay was performed using BD BioCoat endothelial cell Migration Assay System (Becton-Dickinson Labware) according to the provider's manual. Briefly, 1610 5 negative siRNA, Prox1 siRNA, or FOXC2 siRNA treated KON cells were seeded in a 24-well plate. After overnight incubated at 37uC, 5610 4 cells of HUVECs or HDLMVECs were seeded in fibronectin pre-coated transwell chambers, consisting of polycarbonate membranes with 8 mm pores and then placed in the 24-well plates. After 24 h incubation, migrating cells were fluorescent labeling and measured intensity of fluorescence.

Statistical Analysis
Statistical analysis was carried out with JMP8 (SAS Institute, Cary, NC, USA). Statistical differentiation was calculated with x 2 test, one-factor ANOVA test, and student-t test. Disease-free survival was analyzed by the Kaplan-Meier method, and differences between groups were calculated by means of a logrank test. Univariate analysis for disease free survival was calculated by logrank test. For multivariate analysis, Cox proportional hazards model was used (described as hazard ratio with 95% confidence intervals [95% CI], together with the P value). P values less than 0.05 were regarded as statistically significant.

Expression of Prox1 and FOXC2 in Human OSCC Specimens
First, we examined the expression of Prox1 and FOXC2 in human OSCCs by immunohistochemistry. Although non-cancerous oral mucosa did not express Prox1 (Fig. 1a) and FOXC2 (Fig. 1b), immunopositivity of Prox1 was observed in lymphoendothelial cells (LECs) (Fig. 1c) and expression of FOXC2 was observed in LECs and vascular endothelial cells (VECs) (Fig. 1d). Nuclear staining for these factors was observed in OSCCs, and 25.8% (42/163) and 23.3% (38/163) of OSCCs were positive for Prox1 (Fig. 1e) and FOXC2 (Fig. 1f), respectively. The relationship between the expression of Prox1 and FOXC2 and the clinicopathological characteristics of the OSCC specimens are summarized in Table 1. Immunoreactivity for Prox1 was observed in 62% (31/50) of the nodal metastasis-positive cases, whereas 9.7% (11/113) of the cases without nodal metastasis expressed Prox1 (P,0.0001). Prox1 expression was also associated with local progression of the tumor (T classification) (P = 0.0023), clinical stage (P,0.0001), and lymphatic vessel density (LVD) (Fig. 1g) (P,0.0001). No significant relationship was observed between the expression levels of Prox1 and age, sex, site, histological differentiation, or microvessel density (MVD). The expression of FOXC2 was associated with MVD alone (Fig. 1h) (P,0.0001). Elevated expression of Prox1 was observed to be correlated with the overexpression of FOXC2 in OSCCs (P,0.0001).
We then assessed the expression of Prox1 and FOXC2 mRNA in 5 samples of non-tumor oral mucosa, 10 samples of metastasisnegative OSCCs, and 5 samples of metastasis-positive OSCCs (Fig. 2). The expression of Prox1 (P,0.01) and FOXC2 (P,0.01) were higher in OSCCs than in the normal oral mucosa. Although Prox1 was upregulated in nodal metastasis-positive OSCCs unlike that in the metastasis-negative OSCCs (P,0.01), the expression levels of FOXC2 were not significantly different between the 2 OSCC groups.

Relation between Prox1 and FOXC2 Expression and Prognosis of OSCCs
Local and nodal recurrence occurred in 38 of the 163 patients whose tumor specimens were evaluated in this study. An analysis of disease-free survival showed that Prox1-positive patients had significantly reduced disease-free survival compared to the Prox1negative patients (P,0.0001) (Fig. 3a). The patients with FOXC2positive tumors also had significantly worse prognosis than those with FOXC2-negative tumors (P = 0.0076) (Fig. 3b). Univariate analysis performed using the log-rank test indicated that clinical stage (P = 0.0055), nodal metastasis (P = 0.0002), Prox1 expression (P,0.0001), and FOXC2 expression (P = 0.0018) were associated with poor outcome in OSCCs. Multivariate analysis performed using the Cox proportional hazards model showed that only Prox1 expression (P = 0.0039) was a prognostic factor for disease-free survival ( Table 2).

In vitro Analysis of Prox1 and FOXC2 in OSCC Cells
We evaluated Prox1 and FOXC2 expression in cultured OSCC cells. Expression levels of Prox1 and FOXC2 in KON cells were higher than that in the other OSCC cell lines (Fig. 4a). To examine the effects of Prox1 and FOXC2 in OSCC, we further performed an in vitro analysis using KON cells. Cell growth and invasion potential of the KON cells treated with Prox1 siRNA was inhibited compared to that of the cells treated with the negative control siRNA. Knockdown of FOXC2 with siRNA did not affect the ability of KON cells to grow and invade (Fig. 4b, c).
Next, we verified the effects of Prox1 and FOXC2 on the expression of the genes encoding for the VEGF family in KON cells, because both factors were associated with MVD or LVD (Fig. 4d). Reduction of VEGF-C was observed following the knockdown of Prox1, whereas VEGF-A expression was attenuated upon treatment with FOXC2-specific siRNA in the KON cells. Furthermore, downregulation of Prox1 was observed in FOXC2 siRNA-treated KON cells. However, reduction of FOXC2 was not observed in KON cells treated with Prox1-specific siRNA. These results suggest that Prox1 was involved in regulating the expression of VEGF-C and that FOXC2 expression accelerated not only the activation of VEGF-A but also of the Prox1 in OSCC cells.
Finally, we confirmed the influence of Prox1 and FOXC2 on angiogenesis and lymphangiogenesis in OSCC cells. The proliferation of HUVECs and HDLMVECs were significantly enhanced by the addition of culture supernatant derived from negative control siRNA-treated KON cell cultures, suggesting that the KON cells secreted cell growth-promoting factors. However, treatment of HUVECs or HDLMVECs with the culture supernatant from FOXC2-or Prox1-specific siRNA-treated KON cell cultures, respectively, resulted in the inhibition of cell growth (Fig. 5a). The migration of HUVECs and HDLMVECs was potentiated by co-culture with negative control siRNA-treated KON cells. However, co-culture with FOXC2-or Prox1-specific siRNA-treated KON cells significantly suppressed the migration of HUVECs or HDLMVECs, respectively, unlike hat observed in the cells that received the control treatment (Fig. 5b). These data pinpoint the roles of Prox1 and FOXC2, as regulators of lymphangiogenesis and angiogenesis in OSCC, respectively. In addition, we obtained similar results in other OSCC cells (data not shown).

Discussion
In the present study, we found that the mRNA expression levels of Prox1 or FOXC2 in OSCCs were higher than that in the normal oral mucosa. By immunohistochemistical analysis, Prox1 was shown to be closely associated with tumor progression (T factor and clinical stage), nodal metastasis, and LVD, and FOXC2 expression was shown to be significantly correlated with MVD in OSCCs. The patients with Prox1-or FOXC2-positive OSCCs had shorter disease-free days, and Prox1 expression was an independent poor prognostic factor in OSCC. We also found that Prox1 accelerates migration, invasion, VEGF-C expression, and lymphovascular endothelial cell proliferation and migration in OSCC cells. Furthermore, FOXC2 was shown to regulate the expression of VEGF-A and Prox1 as well as the growth and migration of vascular endothelial cells. We also confirmed that Prox1 and FOXC2 induced tube formation in the endothelial cells (data not shown). We speculate that the interactions of Prox1 and VEGF-C are the cause of reduce growth and migration ability of HUVECs by Prox1 knockdown treatment (Fig. 4d, Fig. 5). Indeed, it has been defined tumor cells-secreted VEGF-C knockdown inhibits HUVECs proliferation and migration [38] and we also clarified VEGF-C is accelerates angiogenesis in OSCC [29]. We also infer that the reason for growth and migration potential of HDLMVECs reduce upon siRNA treatement of FOXC2 are FOXC2 regulates expression of Prox1 (Fig. 4d, Fig. 5).
Prox1 is a nuclear transcription factor [22] and is activated in colon cancer [11,19], WHO grade II gliomas [10], and many vascular endothelial tumors [20,21]. Prox1 has been reported to promote tumor progression by influencing cancer cell migration and invasion in colon cancer [19] and kaposiform hemangioendothelioma [20], and our results are consistent with these findings. Elsir et al indicate that enhanced expression of Prox1 in the context of activated Wnt/b-catenin signaling and loss of p53 function may be associated with oncogenesis [22]. On the other hand, 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]. It has been revealed that expression of Angiopoietin 2 (Ang2) is enhanced by Prox1 in endothelial cells [39]. Further, Ang2 is one of the important regulators of angiogenesis and lymphangiogenesis and correlated with poor prognosis in OSCC [40]. Prox1 may play a critical role in the Ang2-induced lymphangiogenesis and angiogenesis of OSCC. Further functional and large scale clinicopathological examinations using various series of cancers are warranted in order to clarify the roles of Prox1 in cancers.
FOXC2 is an oncogene in breast cancer [25], colon cancer [26], and esophageal cancer [27]. Although FOXC2 protein has a cytoplasmic localization in cancer cells [25][26][27], our immunohistochemistry results showed that FOXC2 is detectable in the nuclei of cancer cells. We also verified that FOXC2 protein cannot be detected by immunoblotting using a fraction containing extranuclear proteins extracted from OSCC cells (data not shown). FOXC2 protein may be transported outside the nucleus in certain types of cancers, and further studies will be needed to delineate the localization of FOXC2 in various cancers. FOXC2 is also one of the key players in the epithelial-to-mesenchymal transition (EMT) [28] and EMT inducers, such as TGF-b, stimulate FOXC2 expression in cancer cells [24]. FOXC2 represses and activates Ecadherin and vimentin expression, respectively [24,28]. We also ascertained that FOXC2 regulates the expression of some EMTrelated markers in OSCC cells (unpublished data); however, further studies are required to ascertain the relationship between FOXC2 and EMT in OSCC.
Angiogenesis plays a critical role in prenatal development, wound healing, chronic inflammation, tumor progression, and metastasis, and lymphangiogenesis promotes growth and nodal metastasis in cancer cells [41]. Prox1 is a regulator of lymphangiogenesis during prenatal development and inflammation through the upregulation of VEGFR3 [22,32], and we also confirmed that Prox1 promotes lymphangiogenesis by activating VEGF-C, the product of which is one of the ligands of VEGFR3, in OSCCs. However, a direct role for Prox1 in angiogenesis has not been established yet. A previous report showed that Prox1 transformed blood endothelial cells to lymphatic endothelial cells [42]. Prox1 produced by cancer cells may also trigger lymphangiogenesis from lymphatic and blood endothelial cells in OSCCs. FOXC2 is pivotal for the migration and tubular transformation of vascular endothelial cells and for tumor angiogenesis, functions that are elicited by the activation of VEGF-A signaling [24,26]. Although FOXC2 is also expressed in lymphatic endothelial cells, the contribution of FOXC2 to lymphangiogenesis in malignancies remains unknown [24,34]. We determined that FOXC2 induces tumor angiogenesis through VEGF-A, but that it is not involved in lymphangiogenesis in OSCCs, results that are somewhat in accord with past reports. It is generally accepted that angiogenesis and/or lymphangiogenesis promote tumor progression in malignancies [41], and we previously reported that VEGF family-mediated angiogenesis and/or lymphangiogenesis are associated with tumor progression, nodal metastasis, and worse prognosis in OSCCs [2,29]. On the other hand, it has been suggested that angiogenesis and lymphangiogenesis do not necessarily promote tumor progression. Previously reports have been indicated that VEGF-A is not associated with angiogenesis and that VEGF-C/D are not associated with nodal metastasis in cancer [43][44][45][46]. It has also been reported that MVD and LVD have no effect on tumor progression [47,48]. Therefore, the role of angiogenesis and lymphangiogenesis in tumor progression remains controversial. In vivo studies will be helpful in the future to further clarify the role of tumor angiogenesis and lymphangiogenesis in malignancies.
In conclusion, the present study demonstrates that Prox1 and FOXC2 act as oncogenes by inducing lymphangiogenesis and angiogenesis in OSCC, respectively. Moreover, we found that FOXC2 is involved in the regulation of Prox1 expression. Further investigation of Prox1 and FOXC2 expression and function may offer additional insights for the diagnosis and treatment of human OSCCs.