ANGPTL3 is a novel biomarker as it activates ERK/MAPK pathway in oral cancer

Angiopoietin-like 3 (ANGPTL3), which is involved in new blood vessel growth and stimulation of mitogen-activated protein kinase (MAPK), is expressed aberrantly in several types of human cancers. However, little is known about the relevance of ANGPTL3 in the behavior of oral squamous cell carcinoma (OSCC). In this study, we evaluated ANGPTL3 mRNA and protein in OSCC-derived cell lines (n = 8) and primary OSCCs (n = 109) and assessed the effect of ANGPTL3 on the biology and function of OSCCs in vitro and in vivo. Significant (P < 0.05) ANGPTL3 upregulation was detected in the cell lines and most primary OSCCs (60%) compared with the normal counterparts. The ANGPTL3 expression level was correlated closely (P < 0.05) with tumoral size. In patients with T3/T4 tumors, the overall survival rate with an ANGPTL3-positive tumor was significantly (P < 0.05) lower than that of ANGPTL3-negative cases. In vitro, cellular growth in ANGPTL3 knockdown cells significantly (P < 0.05) decreased with inactivated extracellular regulated kinase (ERK) and cell-cycle arrest at the G1 phase resulting from upregulation of the cyclin-dependent kinase inhibitors, including p21Cip1 and p27Kip1. We also observed a marked (P < 0.05) reduction in the growth in ANGPTL3 knockdown-cell xenografts with decreased levels of phosphorylated ERK relative to control-cell xenografts. The current data indicated that ANGPTL3 may play a role in OSCCs via MAPK signaling cascades, making it a potentially useful diagnostic/therapeutic target for use in patients with OSCC.


Introduction
Angiopoietin-like 3 (ANGPTL3) is a member of the angiopoietin-like proteins (ANGPTLs), which are functionally defined by the C-terminal fibrinogen-like domain that mediates binding to the Tie2 receptor and thereby facilitates a cascade of ultimate events regulating blood vessel formation [1][2][3]. However, ANGPTL3 binds to integrins and not the Tie 2 receptor [1].
ANGPTL3 plays a vital role in regulating the plasma levels of triglyceride and cholesterol mainly via reversible inhibition of the lipoprotein lipase activity and vascular endothelial growth factors [2,3].
Previous studies have reported that ANGPTL3 activity is one of the most important factors in cancer growth and invasion [4], because of the mitogen-activated protein kinase (MAPK) signaling cascades [5]. Growth of human cancer cells is controlled largely at the G1 phase of the cell cycle [6]. ANGPLT3 was reported that specifically modulates upstream of the extracellular-regulated kinase (ERK)/MAPK signaling cascade by a previous study [7]. The activation of ERK plays a fundamental role in G1/S transition [8,9]. Protein/kinase inhibitory protein (Cip/Kip) family binds to cyclin-CDK complexes interacting to the members of the cyclin-dependent kinase and leads to G1 cell-cycle arrest. The levels of p21, p27, cyclin D1, and cyclin E are influenced by multiple signaling pathways including the (ERK)/MAPK signaling pathway [10][11][12][13]. Based on this evidence, we hypothesized that ANGPTL3 may be associated with development of human cancer including oral squamous cell carcinoma (OSCCs).
This study shows the results of a comprehensive analysis of molecular/cellular subtypes of ANGPTL3 in OSCC that are linked clinically to tumoral progression and prognosis.

Ethics statement
The study protocol was approved by the Ethical Committee of the Graduate School of Medicine, Chiba University (approval number, 236) and performed in accordance with the tenets of the Declaration of Helsinki. All patients provided written informed consent before participating in this research.
The care of the animals was in accordance with the guidelines of Chiba University. The Committee on the Ethics of Animal Experiments of Chiba University approved the study protocol (approval number, 25-221).

Immunohistochemistry
Immunohistochemistry and IHC scoring systems were performed as described previously [14][15][16][17]. The antibodies were anti-ANGPTL3 (Santa Cruz Biotechnology). To calculate the 5-year survival rate, we surveyed each patient's life and month of death.

Transfection with shRNA plasmid
Stable transfection was performed using Lipofectamine LTX and Plus Reagents (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions. ANGPTL3 shRNA (shANGPTL3) and the control shRNA (shMock) (Santa Cruz) vectors were transfected into HSC-3 and Sa3. After transfection, the cells were isolated by the culture medium containing 1 mg/mL Puromycin (Invitrogen). After 3-4 weeks, resistant cell colonies were picked and transferred to new dishes. ShANGPTL3-and shMocktransfected cells were used for further experiments.

Cell-cycle analysis
In order to synchronize cells at the G0/G1 or G2/M transition, they were deprived of serum for 48 h or treated with 200 ng/mL nocodazole (Sigma, St. Louis, MO, USA) for 20 h according to the previous reports [18,19]. Cellcycle analysis was performed as described previously [14][15][16][17].

Tumorigenesis and tumoral growth in vivo
To investigate whether ANGPTL3 expression contributed to tumorigenesis and tumoral growth, we used xenograft models in two cell lines (HSC-3 and Sa3). The cells (2 9 10 7 ) were independently injected subcutaneously into the backs of female nude mice, BALB/c-nu, purchased from Oriental Yeast Co. (Tokyo, Japan). All experimental animals were treated and cared for in accordance with institutional guidelines. The tumoral sizes were measured using a digital caliper every 3-4 days after injection, when the volume of the transplantation tumor reached 100 mm 3 . We used the formula 4p/3 9 (width/2) 2 9 (length/2) to calculate tumoral volume. The mice were sacrificed after 28 days. Tumor tissues were fixed in 10% formalin, and paraffin sections (4 lm) were prepared for hematoxylin and eosin staining and IHC of anti-ANG-PTL3, anti-ERK1/2, anti-pERK1/2, and anti-Ki-67 (Santa Cruz Biotechnology).

Statistical analysis
In comparisons of ANGPTL3 expression levels, statistical significance was evaluated using the Mann-Whitney Utest. Relationships between ANGPTL3 IHC staining scores and clinicopathological profiles were evaluated using the v 2 test, Fisher's exact test, and Mann-Whitney U-test. Survival curves were obtained using the Kaplan-Meier method, and differences in survival rates between ANGPTL3-positive and -negative cases were compared using the log-rank test. P < 0.05 was considered significant. The data are expressed as the mean AE standard error of the mean (SEM). We then performed the receiver operating characteristic (ROC) curve analysis by plotting sensitivity versus specificity, and area under the ROC curve (AUC) values with estimate odds ratios and 95% confidence intervals (CIs) as described previously with a slight modification [20]. The identified data were expressed as the mean AE SEM. Statistical analyses were performed using EZR (Saitama Medical Center, Jichi Medical University, accessed 1 March 2012, at http:// www.jichi.ac.jp/saitama-sct/SaitamaHP.files/statmedENstat medEN.html), which is a graphical user interface for R (The R Foundation for Statistical Computing, version 2.13.0) [21].

Evaluation of ANGPTL3 expression in primary OSCCs
We analyzed the ANGPTL3 protein expression in primary OSCCs from 109 patients using an IHC scoring system. The ANGPTL3 IHC scores in OSCCs and adjacent normal oral tissues ranged from 55.5 to 200.0 (median, 138.6) and 30.5 to 105.2 (median, 65.5), respectively. Representative IHC results for ANGPTL3 protein in adjacent normal oral tissue and primary OSCCs are shown in Figure 2A and B, respectively. Strong ANGPTL3 immunoreactivity was detected in the cytoplasmic of OSCC tis- sues, whereas the normal tissues showed almost negative immunostaining. The IHC scores in primary OSCCs were significantly (P < 0.05) higher than in normal oral tissues (Fig. 2C). The correlations between the clinicopathological characteristics of the patients with OSCC and the status of ANGPTL3 protein expression are shown in Table 1. Among the clinical parameters, the ANGPTL3 expression level was significantly (P < 0.05) related to the primary tumoral size of the OSCCs. Survival analysis using the Kaplan-Meier method showed that the ANG-PTL3 expression level was not significantly (P = 0.076) correlated with overall survival (Fig. 2D). The overall survival rates in the ANGPTL3-positive OSCCs (n = 65) and the ANGPTL3-negative OSCCs (n = 44) were 78.7% and 89.9%, respectively. However, a significant (P = 0.047) difference was found in overall survival rates of patients with primary T3/T4 OSCC tumors between the ANGPTL3-positive OSCCs (n = 31; 58.6%) and the ANGPTL3-negative OSCCs (n = 12; 91.6%) (Fig. 2E). Furthermore, the diagnostic accuracy of the identified IHC scores was assessed using the ROC curve analysis. The ANGPTL3 IHC scores of patients with primary T3/ T4 OSCC tumors in primary OSCCs normalized to those in normal tissues. The optimal threshold value was 2.25 (sensitivity, 76.9%; specificity, 69.2%). When the cutoff values for the ANGPTL3 IHC scores were set at 2.25, the AUC was 0.7293 (95% CI, 0.5682-0.8903, P < 0.05) (Fig. 2F).

Establishment of ANGPTL3 knockdown cells
Since frequent upregulation of ANGPTL3 was observed in OSCC-derived cells (Fig. 1), the OSCC-derived cells (HSC-3 and Sa3) were transfected with ANGPTL3 shRNA and shMock as controls. To confirm that shANGPTL3 transfection works and ANGPTL3 mRNA and protein decrease, we performed qRT-PCR and Western blotting ( Fig. 3A and B, respectively). The ANG-PTL3 mRNA expression in shANGPTL3 cells was significantly (P < 0.05) lower than in shMock cells (Fig. 3A). The ANGPTL3 protein level in the shANG-PTL3 cells also was decreased compared with shMock cells (Fig. 3B).

Functional analyses of ANGPTL3 knockdown cells
We performed a cellular proliferation assay (Fig. 3C) to evaluate the effect of ANGPTL3 knockdown on cellular growth, we found a significant (P < 0.05) decrease in cellular growth in all shANGPTL3 cells compared with shMock cells. The assays showed that ANGPTL3 knockdown decreased cellular growth.

Inactivation of the ERK pathway in shANGPTL3 cells
We assessed the phosphorylation level of ERK in shANG-PTL3 by immunoblotting analysis. The level of phosphorylated ERK (pERK) protein decreased significantly in shANGPTL3 cells compared with shMock cells (Fig. 3D). Furthermore, to evaluate the effect of ERK inhibitor, we performed immunoblotting analysis with 10 lmol/L PD184352 (Sigma) or an equivalent 0.1% DMSO (control) for 90 min [22,23]. Results from immunoblotting analysis indicated the similar results in shANGPTL3 cells (Fig. S1A). These results suggested that the ERK signaling pathway was attenuated frequently in the shANGPTL3 cells.

Cell-cycle analysis of shANGPTL3 cells
We assessed the expression levels of the cyclin-dependent kinase inhibitors (CDKIs) (p21 Cip1 and p27 Kip1 ), cyclins, and CDKs. As expected, the CDKIs were upregulated, and cyclin D1, cyclin E, CDK2, CDK4, and CDK6 were significantly (P < 0.05) downregulated in the shANGPTL3 cells (Fig. 3E). Moreover, the percentage of the shANGPTL3 cells at the G1 phase was significantly (P < 0.05) higher than of the Mock cells (Fig. 3F). These results indicated that shANGPTL3 inhibited cellular proliferation by cellcycle arrest at the G1 phase.

ANGPTL3 promoted tumoral growth in vivo
We assessed the effect of ANGPTL3 on tumoral growth in vivo by evaluating to target tumor xenografts in nude mice. shANGPTL3-and shMock-transfected cells of two cell lines, HSC-3 and Sa3 were injected subcutaneously into the backs of female nude mice, respectively (three mice in each group). According to our in vitro findings, the mean tumoral volume of the shANGPTL3-transfected cells was significantly (P < 0.05) smaller than that of the shMock-transfected cells (Fig. 4A). ANGPTL3 IHC of tumoral sections showed that ANGPTL3 knockdown was maintained in vivo. Xenografted tumors of ANGPTL3 knockdown cells showed a significant decrease in the pERK and Ki-67 levels ( Fig. 4B and Fig. S1B). However, the ERK levels were unchanged. These study provided that ANGPTL3 promotes tumoral growth by the ERK pathway in nude mice.

Discussion
We showed that ANGPTL3 was overexpressed frequently in all OSCC-derived cell lines examined (Fig. 1). The knockdown of ANGPTL3 in OSCC-derived cell lines resulted in a dramatic effect on growth inhibition in vitro and in vivo via arrest of the G1/S phase by inactivation of ERK with upregulation of CDKI including p21 Cip1 and p 27 Kip1 , suggesting that ANGPTL3 may predict tumoral progression and be a prognostic factor of this disease. (Figs. 3 and 4). The expression and function of ANGTPL3 in OSCCs have not been investigated previously. In this study, we found that ANGPTL3 was upregulated at the pretranscription and protein levels in all OSCC-derived cell lines examined (Fig. 1). To investigate the possible function of ANGPTL3, we created stable ANGPTL3 knockdown transfectants in two OSCC-derived cell lines ( Fig. 3A and B). Interestingly, suppression of ANGPTL3 significantly decreased cellular proliferation (Fig. 3C), which suggested the hypothesis that ANGPTL3 expression is necessary for oral carcinogenesis and neoplastic progression.
It is thought that carcinogenesis of human cancers including OSCC are multistep processes with epithelial cellular proliferation of progressive disruption. The study of cellular proliferation mechanism in tumoral cells is important for tumoral treatment and molecular biology [24,25]. ANGPTL3 has been reported to be associated with tumoral development and progression [26]. In this study, suppression of ANGPTL3 by shRNA significantly decreased cellular proliferation by inhibiting the ERK pathway (Fig. 3D). Knockdown of ANGPTL3 is related closely to hyperactivation of the ERK signaling pathway, which is linked to fundamental cellular processes such as  cell-cycle arrest and proliferation [27,28]. In addition, extracellular signals tightly control the ERK pathway [29][30][31], which include the ANGPTL3 binding to integrin a c b3 [1,7,32]. Based on this evidence combined with the current results, we believe that ANGPTL3 may be correlated at least indirectly with aberrant activation of ERK/ MAPK signaling pathways. More importantly, our OSCC cells with shANGPTL3 have the characteristics to arrest with upregulation of p21 Cip1 and p 27 Kip1 and downregulation of cyclin D1 and cyclin E at the G1 phase of cell cycle (Fig. 3E). Considerable evidence has shown that while cyclin D1 and cyclin E are upregulated [14,33], the CDKIs including p21 Cip1 and p 27 Kip1 are downregulated in human oral cancers [15,16]. In addition, G1-S transition of cell cycle is blocked by dramatic inhibition of their expression [9,34,35]. Members of the Cip/Kip family, p21 Cip1 and p27 Kip1 , are implicated in the negative regulation of cellcycle progression from the G1 to S phase by binding to and modulating CDK activity [8,[36][37][38][39]. In contrast, CDK complexes promote progression from the G1 to S phase by triggering DNA replication and regulating genes [35,40,41]. ERK regulates p21 Cip1 and p27 Kip1 negatively and CDK complexes positively [35,40,41]. These data suggested that ANGPTL3 regulates the ERK signaling pathway and promotes the G1 cell cycle during OSCC progression.
Moreover, to investigate the oncogenic potential of ANGPTL3 in vivo, we tested a xenograft assay. Our in vivo data showed significant inhibition of xenografted tumoral growth and transformation, and activation of ERK in tumoral tissues by ANGPTL3 silencing. Therefore, our current study suggested that ANGPTL3 activates the MAPK signaling pathway via pERK and promotes the G1 cell cycle by downregulating CDKIs during OSCC progression.
Based on our in vivo and in vitro data, we concluded that aberrant ANGPTL3 expression in primary OSCCs may contribute to neoplastic promote via the ERK pathway that ANGPTL3 may play an important role in OSCC cellular growth. It would be interesting to design clinical studies to elucidate the predictive relevance of ANGPTL3 expression status in future patients with OSCC, because advanced OSCC cases (T3/T4) have been divided into two groups, those with favorable and unfavorable prognoses (Fig. 2 E and F), based on the ANGPTL3 status in primary OSCCs.