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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.

ABSTRACT: 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).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.mRNA and protein expression of NUSAP1 were significantly up-regulated in OSCC-derived cells compared with human normal oral keratinocytes (P < 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 (P < 0.05) inhibition of cellular proliferation. Furthermore, apoptosis induced by PTX was enhanced in NUSAP1 knockdown OSCC cells.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.

SUBMITTER: Okamoto A

PROVIDER: S-EPMC4640814 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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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.

Okamoto Atsushi A Higo Morihiro M Shiiba Masashi M Nakashima Dai D Koyama Tomoyoshi T Miyamoto Isao I Kasama Hiroki H Kasamatsu Atsushi A Ogawara Katsunori K Yokoe Hidetaka H Tanzawa Hideki H Uzawa Katsuhiro K

PloS one 20151110 11

<h4>Background</h4>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).<h4>Methods</h4>mRNA and protein expression levels of NUSAP1 in 9 OSCC-derived cells were analyzed by quantitative reverse transcriptase-polymerase chai ...[more]

PMID: 26554377

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Project description:BackgroundPaclitaxel (PTX), a first-line therapy for triple negative breast cancers (TNBC) induces anti-tumor activity by microtubule stabilization and inhibition of cell division. Its dose-limiting toxicity and short half-life, however, pose clinical challenges underscoring the need for strategies that increase its efficiency. RAD6, a E2 ubiquitin conjugating enzyme, is associated with centrosomes at all phases of cell cycle. Constitutive overexpression of the RAD6B homolog in normal breast cells induces centrosome amplification and multipolar spindle formation, indicating its importance in centrosome regulation.MethodsTNBC centrosome numbers were scored by pericentrin immunostaining. PTX sensitivities and interactions with SMI#9, a RAD6-selective small molecule inhibitor, on TNBC cell survival were analyzed by MTT and colony forming assays and an isogenic MDA-MB-468 TNBC model of PTX resistance. The molecular mechanisms underlying PTX and SMI#9 induced cytotoxicity were determined by flow cytometry, immunoblot analysis of cyclin B1 and microtubule associated protein TAU, and dual immunofluorescence staining of TAU and α-tubulin.ResultsOur data show aberrant centrosome numbers and that PTX sensitivities are not correlated with TNBC BRCA1 status. Combining PTX with SMI#9 synergistically enhances PTX sensitivities of BRCA1 wild-type and mutant TNBC cells. Whereas SMI#9/PTX combination treatment increased cyclin B1 levels in MDA-MB-468 cells, it induced cyclin B1 loss in HCC1937 cells with accumulation of reproductively dead giant cells, a characteristic of mitotic catastrophe. Cell cycle analysis revealed drug-induced accumulation of tetraploid cells in S and G2/M phases, and robust increases in cells with 4 N DNA content in HCC1937 cells. TAU overexpression is associated with reduced PTX efficacy. Among the six TAU isoforms, both SMI#9 and PTX downregulated 1N3R TAU in MDA-MB-468 and HCC1937 cells, suggesting a common mechanism of 1N3R regulation. Dual TAU and α-tubulin immunostaining showed that SMI#9 induces monopolar mitotic spindles. Using the isogenic model of PTX resistance, we show that SMI#9 treatment restores PTX sensitivity.ConclusionsThese data support a common mechanism of microtubule regulation by SMI#9 and PTX and suggest that combining PTX with RAD6 inhibitor may be beneficial for increasing TNBC sensitivities to PTX and alleviating toxicity. This study demonstrates a new role for RAD6 in regulating microtubule dynamics.

Dataset Information

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.

Publications

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.

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