Down-Regulation of Nucleolar and Spindle-Associated Protein 1 (NUSAP1) Expression Suppresses Tumor and Cell Proliferation and Enhances Anti-Tumor Effect of Paclitaxel in Oral Squamous Cell Carcinoma

<div><p>Background</p><p>Nucleolar and spindle-associated protein 1 (NUSAP1) is an important mitotic regulator. In addition to its crucial function in mitosis, NUSAP1 has recently received attention due to the interesting roles in carcinogenesis. The aim of this study was to reveal functional mechanisms of NUSAP1 in oral squamous cell carcinoma (OSCC).</p><p>Methods</p><p>mRNA and protein expression levels of NUSAP1 in 9 OSCC-derived cells were analyzed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and immunoblotting analyses. The correlation between the NUSAP1 expression profile and the clinicopathological factors was evaluated by immunohistochemistry (IHC) in clinical OSCC samples (n = 70). The NUSAP1 knockdown cells were established with short hairpin RNA (shRNA) in OSCC cells, and functional assays were performed using these cells. In addition to the evaluation of cellular proliferation and cell cycle, we also investigated the potential role of NUSAP1 in paclitaxel (PTX)-induced cellular responses.</p><p>Results</p><p>mRNA and protein expression of NUSAP1 were significantly up-regulated in OSCC-derived cells compared with human normal oral keratinocytes (<i>P</i> < 0.05). IHC revealed that NUSAP-1 expression is closely associated with primary advanced T stage (P<0.05). Suppression of NUSAP1 expression levels led to significant (<i>P</i> < 0.05) inhibition of cellular proliferation. Furthermore, apoptosis induced by PTX was enhanced in NUSAP1 knockdown OSCC cells.</p><p>Conclusions</p><p>NUSAP1 may be a crucial biomarker for OSCC. Moreover, down-regulated NUSAP1 expression suppresses tumor proliferation and also enhances anti-tumor effect of PTX by activating apoptotic pathways. Thus, the present study strongly suggests that regulating NUSAP1 expression should contribute to the therapy for OSCC.</p></div

Evaluation of NUSAP1 protein expression in primary OSCCs.

<p>Representative IHC results for NUSAP1 in a normal oral tissue and b primary OSCC (original magnification, x400. Scale bars, 50 μm). Strong NUSAP1 immunoreactivity is detected in primary OSCCs, whereas normal oral tissues show almost negative immunostaining. c The state of NUSAP1 protein expression in the normal counterparts and primary OSCCs (n = 70). The NUSAP1 IHC scores for normal oral tissues and OSCCs range from 0 to 38.1 (median 12.8) and 40.2 to 170.3 (median 136.3), respectively. NUSAP1 protein expression levels in OSCCs are significantly (<i>p</i> < 0.001, Mann-Whitney’s <i>U</i> test) higher than that in normal oral tissues.</p

Functional analyses of NUSAP1 knockdown cells.

<p>Reduced cellular growth was observed in a shNUSAP1 HSC-3 and b shNUSAP1 Sa3 cells. To determine the effect of shNUSAP1 on cellular proliferation, shNUSAP1 and Mock cells are seeded in 6-well plates at a density of 1×10⁴ viable cells/well. The shNUSAP1 HSC-3 and shNUSAP1 Sa3 cells show a significant (<i>p</i> < 0.05, Mann-Whitney’s <i>U</i> test*) decrease in cellular growth compared with Mock cells. The results are expressed as the means ± SEM of values from three assays. The asterisks indicate significant (<i>p</i> < 0.05, Mann-Whitney <i>U</i> test*) differences between the shNUSAP1 and Mock cells. c shNUSAP1- and Mock-transfected cells were counted on 4 consecutive days by a hemocytometer.</p

Evaluation of NUSAP1 expression in OSCC-derived cell lines.

<p>a. Quantification of <i>NUSAP1</i> mRNA expression levels in OSCC-derived cell lines by qRT-PCR analysis. mRNA expression levels are normalized to GAPDH. Significant (<i>p</i> < 0.05, Mann-Whitney’s <i>U</i> test*) up-regulation of <i>NUSAP1</i> mRNA is seen in nine OSCC-derived cell lines (HSC-2, HSC-3, HSC-4, KOSC2, Ca9-22, Sa3, HO-1-N-1, HO-1-u-1, and KON) compared with the HNOKs. Data are expressed as the means ± SEM of triplicate results. b. Representative immunoblotting data of NUSAP1 in OSCC-derived cell lines and HNOKs show that NUSAP1 protein expression is up-regulated in OSCC-derived cell lines compared with the HNOKs. The molecular weight of the NUSAP1 is 49 kDa. Densitometric NUSAP1 protein data are normalized to GAPDH protein levels. The values are expressed as a percentage of the HNOKs.</p

Correlation between NUSAP1 expression and parameters in OSCCs.

<p>NUSAP1(-), down-regulated NUSAP1; NUSAP1(+), up-regulated NUSAP1.</p><p>*<i>p</i> < 0.05. (χ2 test).</p><p>Correlation between NUSAP1 expression and parameters in OSCCs.</p