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

Abstract

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 p21(Cip1) and p27(Kip1) . 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.

Keywords: Angiopoietin-like 3; extracellular signal-regulated MAP Kinases; head and neck; squamous cell carcinoma; survival.

Figure 1

Evaluation of ANGPTL3 expression in OSCC-derived cell lines. (A) Quantification of ANGPTL3 mRNA expression in OSCC-derived cell lines by qRT-PCR analysis. Significant (*P < 0.05, Mann–Whitney U-test) upregulation of ANGPTL3 mRNA is seen in seven OSCC-derived cell lines compared with the HNOKs. Data are expressed as the mean ± SEM of triplicate results. (B) Immunoblotting analysis of ANGPTL3 protein in the OSCC-derived cell lines and HNOKs. ANGPTL3 protein expression is upregulated in the OSCC-derived cell lines compared with the HNOKs. Densitometric ANGPTL3 protein data are normalized to GAPDH protein levels. The values are expressed as a percentage of the HNOKs (P < 0.05, Mann–Whitney U-test).

Figure 2

Evaluation of ANGPTL3 protein expression in primary OSCCs. (A and B) Representative IHC results of ANGPTL3 in primary OSCCs and normal oral tissues. Original magnification, ×100. Scale bars, 50 μm. ANGPTL3 is highly overexpressed in OSCCs compared to normal oral tissues. (C) The status of ANGPTL3 protein expression in primary OSCCs (n = 109) and normal counterparts based on an IHC scoring system. IHC scores are calculated as follows: IHC score = 1 × (number of weakly stained cells in the field) + 2 × (number of moderately stained cells in the field) + 3 × (number of intensely stained cells in the field). The ANGPTL3 IHC scores for normal oral tissues and OSCCs range from 30.5 to 105.2 (median, 65.5) and 55.5 to 200.0 (median, 138.6), respectively. ANGPTL3 protein expression levels in OSCCs are significantly higher than in normal oral tissues (*P = 0.003; Mann–Whitney U test). (D) Kaplan–Meier curve for overall survival. The ANGPTL3 expression level is not correlated significantly (P = 0.076, log-rank test) with the overall survival. 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. (E) Kaplan–Meier overall survival curves of patients with OSCC with a primary tumor size of T3/T4. A significant (P = 0.047, log-rank test) difference is seen in the overall survival rates between the ANGPTL3-positive OSCCs (n = 31, 58.6%) and the ANGPTL3-negative OSCCs (n = 12, 91.6%). (F) To evaluate the diagnostic relevance of the identified IHC scores, we used the ROC curve by plotting sensitivity versus specificity. 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, 75.8%; specificity, 68.2%). 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).

Figure 3

Expression of ANGPTL3 and ANGPTL3 knockdown inhibits ERK activation and promotes G1 arrest. (A) qRT-PCR shows that ANGPTL3 mRNA expression in the shANGPTL3-transfected cells (HSC-3- and Sa3-derived transfectants) is significantly (*P < 0.05, Mann–Whitney U-test) lower than in the shMock-transfected cells. (B) Immunoblotting analysis shows that the ANGPTL3 protein levels in the shANGPTL3-transfected cells (HSC-3- and Sa3-derived transfectants) also are decreased markedly compared with the shMock-transfected cells. (C) To determine the effect of shANGPTL3 on cellular proliferation, shANGPTL3-, and shMock-transfected cells were seeded in six-well plates at a density of 1 × 10⁴ viable cells/well. Both transfectants were counted on seven consecutive days. The cellular growth of shANGPTL3-transfected cells (HSC-3- and Sa3-derived transfectants) is significantly inhibited compared with the shMock-transfected cells after 7 days (168 h). The results are expressed as the means ± SEM of values from three assays. The asterisks indicate significant (*P < 0.05, Mann–Whitney U-test) differences between the shANGPTL3 and shMock cells. (D) Immunoblotting analysis shows that ANGPTL3 knockdown results in decreased levels of pERK compared with the shMock-transfected cells (HSC-3- and Sa3-derived transfectants). Densitometric pERK/ERK protein data are normalized to GAPDH protein levels. (E) Immunoblotting analysis shows upregulation of p21^Cip1 and p27^Kip1 and downregulation of cyclin D1, cyclin E, CDK2, CDK4, and CDK6 in the shANGPTL3-transfected cells (HSC-3- and Sa3-derived transfectants) compared with the shMock-transfected cells. (F) Flow cytometric analysis was performed to investigate cell-cycle progression in the shANGPTL3- and shMock-transfected cells after synchronization at the G2/M phase to treatment with nocodazole. The percentage of cells at the G1 phase in the shANGPTL3-transfected cells (HSC-3- and Sa3-derived transfectants) is increased markedly compared with the shMock- transfected cells.

Figure 4

ANGPTL3 promotes tumoral growth in vivo. (A) ShANGPTL3- and shMock-transfected cells (HSC-3 and Sa3) were injected subcutaneously into the backs of female nude mice (n = 3). Tumoral growth in the shANGTPL3-injected mice is inhibited significantly (*P < 0.05; Mann–Whitney U-test) compared to the shMock-injected mice. (B) IHC of the xenografted tumors clearly shows more decreased immunostaining for ANGPTL3 and pERK in the xenografted tumors from shANGPTL3 transfectants than shMock transfectants. H&E staining confirmed the presence of tumoral cells. Original magnification, ×400. Scale bars, 50 μm.