Nucleostemin (NS) has been reported as essential for stem and cancer cell proliferation. To investigate the significance of NS in oral squamous cell carcinomas (OSCCs), we examined NS expression in neoplastic tissue of the tongue and in OSCC cell lines. Nucleostemin expression in the histological samples showed positive correlation with Ki-67 expression. Furthermore, NS expression was associated with cellular proliferation in OSCC cell lines using siRNA, which upregulated p27, a cyclin-dependent kinase inhibitor. Regarding OSCC differentiation, NS expression did not influence cornification or oral epithelial differentiation markers such as involucrin and cytokeratin19. Thus, NS is widely expressed in normal and neoplastic oral epithelial tissues, and is likely a marker of proliferation. (Cancer Sci 2011; 102: 1418–1423)
Oral cancer is one of the most common forms of cancer.(1,2) The survival rate for patients with oral cancer has not yet improved, despite better diagnostic techniques and innovations in treatments.(3) It is thought that most, if not all, oral squamous cell carcinomas (OSCCs) are preceded by a period during which the affected epithelium shows evidence of epithelial dysplasia, although this may not always be clinically apparent.(4) Histological criteria to assess cellular and tissue changes (dysplasia) have been defined by the World Health Organization(5) and are related to alterations in the kinetics of cellular proliferation and maturation of the epithelium.(6,7) A better understanding of the molecular mechanisms underlying the development of OSCC could add to the treatment and prognosis of this disease.
Nucleostemin (NS) is a newly characterized nucleolar protein, that has been reported to interact with the tumor suppressor protein p53 and to regulate cell proliferation.(8) Several groups have reported the expression of NS in different types of stem cell as well as in several cancer cell lines.(9–13) Both depletion and overexpression of this protein result in cell cycle arrest through p53-dependent and p53-independent mechanisms.(14–16)
Nucleostemin expression is generally downregulated in the early stages of differentiation before exit from the cell cycle.(8,12,17) In addition, knockdown of NS significantly inhibits the proliferation of stem cells and cancer cells.(10,15,18–22) These initial observations led to suggestions that NS is involved in multipotency in stem cells as well as in the regulation of cancer and stem cell proliferation. Recent studies, however, have indicated that NS is in fact widely expressed in many types of normal proliferating cells at levels similar to those in malignant cells.(23–25) However, it is unknown whether NS is involved in the pathogenesis of OSCC.
In the present study, we examined immunohistochemical patterns of NS in the normal epithelium, premalignant lesions (dysplasia), and cancer of the tongue to know the significance of NS in tongue cancer development. Next, we used NS-specific, siRNA to knockdown the NS gene in OSCC cell lines and clarify the involvement of NS in proliferation. Finally, we examined the effect of NS expression on the expression levels of oral epithelial differentiation markers using cornification and Western blot analysis.
Materials and Methods
Patients and tissue specimens. Tissue samples of tongue squamous cell carcinoma (TSCC; n = 10), dysplasia (n = 25), and normal tongue (n = 10) were obtained from 45 patients. The samples were fixed with 10% formalin and embedded in paraffin. Tissues sets were stained with H&E. Histological diagnosis of the samples was made basically according to criteria of the World Health Organization.(5) Besides, we took into consideration the existence of differentiated type dysplasia.(26) These cases were not included in this study. Additional sections were used for immunohistochemistry.
The study followed the guidelines of the Ethics Committee of Kumamoto University (Kumamoto, Japan). The nature and aims of the study were explained to all patients, who gave their informed consent for the research.
Immunohistochemical staining and evaluation. Formalin-fixed paraffin-embedded specimens were cut into 4-μm sections and mounted on MAS-GP-coated slides (Matsunami Glass Industries, Osaka, Japan). After deparaffinization and rehydration, the sections were heated with an autoclave in 0.01 mol/L citrate buffer (pH 7.0) for 15 min at 121°C for antigen retrieval. The sections were incubated with 0.3% H2O2 in absolute methanol for 30 min to block endogeneous peroxidase activity, then incubated with 5% normal goat serum for 30 min to block non-specific staining. The sections were incubated with antibodies against NS (R&D Systems, Abingdon, UK), Ki-67 (MIB-1; Dako, Glostrup, Denmark) at 4°C overnight. This was followed by sequential 60 min incubations with secondary antibodies (EnVision+ System/HRP labeled polymer; Dako), and visualization with the Liquid DAB+ Substrate Chromogen System (Dako). All slides were lightly counterstained with hematoxylin for 30 s prior to dehydration and mounting. Ki-67 was considered positive when cells displayed nuclear positive staining. Nucleostemin was considered positive when cells showed nucleolar staining. The number of positively stained cells out of 100 in five random fields (400 × objective) was counted. A labeling index (LI) was calculated by dividing the number of positive cells by the total number of cells per case and multiplying by 100. Slides were independently reviewed by two pathologists and a consensus was reached.
Cell lines. All human OSCC cell lines, namely, HSC-2, HSC-3, SAS, KB, and Ca9-22, were kindly donated by the Department of Oral and Maxillofacial Surgery, Graduate School of Dental Science, Kyushu University (Maidashi, Higashi-ku, Fukuoka, Japan). All cell lines were cultured in DMEM (Gibco, Grand Island, NY, USA) with 10% FBS. The cells were incubated at 37°C in 5% CO2 and saturated humidity.
Western blot analysis. Cells were lysed on ice in 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA-2Na (pH 8.0), 1 mM EGTA (pH 7.5), 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na2VO4, 1 mM PMSF, and 20 mM Tris-HCl (pH 7.5) containing a protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland). Protein concentrations were determined using the Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). Samples were boiled with Laemmli buffer. Equal amounts of protein were electrophoresed on polyacrylamide gels containing 0.1% SDS, transferred to PVDF membranes (Millipore, Hamburg, Germany), and incubated with specific primary antibodies. Proteins of interest were detected with appropriate HRP-conjugated secondary antibodies and enhanced chemiluminescence substrate (Amersham Pharmacia Biotech, Little Chalfont, UK). The antibodies used are provided in Table S1.
Reverse transcription–polymerase chain reaction. Total RNA was isolated using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the instructions provided by the manufacturer, and reverse transcribed into cDNA using SuperscriptIII (Invitrogen) and oligo(dT). The cDNA was then amplified by PCR with specific primers. The primer sequences used are shown in Table S2. The PCR products were analyzed by agarose gel electrophoresis and visualized by staining with ethidium bromide.
Immunofluorescence microscopy. The cells were fixed in a 4% PBS–paraformaldehyde solution for 30 min and permeabilized with 0.1% Triton X-100 in PBS for 15 min. After blocking with 5% BSA in PBS for 30 min, coverslips were inverted onto a 50 μL drop of the primary antibodies diluted in PBS containing 0.1% Triton X-100 and 5% BSA (R&D Systems, Minneapolis, MN, USA). After 24 h incubation with antibodies and subsequent washing with PBS × 3 (5 min each), coverslips were inverted again onto 50 μL drops of secondary antibodies diluted in PBS containing 5% BSA and 0.1% Triton X-100 (Alexa Fluor 568 donkey anti-goat IgG; Molecular Probes, Eugene, OR, USA). After washing, coverslips were stained with DAPI (Sigma Aldrich, St. Louis, MO, USA) for 5 min, washed with PBS × 3 (5 min each), mounted in Gel Mount Aqueous Mounting Medium (Sigma Aldrich) and examined by fluorescent microscope.
Small interfering RNA knockdown. SAS cells, with the wild-type TP53 cell line,(27) and Ca9-22 cells, with a mutant TP53cell line,(28) were used in this experiment. Twenty-four hours before transfection, SAS and Ca9-22 cells were diluted in fresh medium without antibiotics and transferred to six-well plates. Cells were grown and transfected with NS or scrambled siRNA (50 nM; Invitrogen) using Lipofectamine RNAi MAX (Invitrogen) as described in manufacturer’s instructions. The sequences for siRNA were described previously.(8) The cells were harvested at 48 h post-transfection for Western blotting.
Cell proliferation analysis. Two thousand SAS cells were cultured in 100 μL of medium per well of a 96-well plate. To examine cell proliferation, an MTT assay was carried out as follows: 10 μL MTT (Nacalai Tesque, Kyoto, Japan) was added to each well 48 h after transfection of NS or scrambled siRNA. The cells were then incubated for 4 h, the medium was replaced with 100 μL DMSO, and the cells were dissociated. Cell proliferation was quantified using a microplate reader (Bio-Rad) with the absorption spectrum set at 570 nm.
Cornification of SAS cells in vitro. SAS cells were cultured in keratinocyte serum-free free-Ca2+ medium (KSFM; Gibco) containing 0.5 mM Ca2+. Control SAS cells were cultured in serum-free free-Ca2+ KSFM containing 0.05 mM Ca2+. The cultures were incubated for 5 days at 37°C, in 5% CO2 and saturated humidity with the medium changed every 2 days. To evaluate the degree of cornification, cultured cells were stained with rhodanile blue according to the method of Rheinwald and Green.(29,30) Both cultures were harvested for Western blotting to examine the changes in the expression of NS and differentiation markers.
Statistical analysis. Statistical analyses were carried out using Statcel2 statistical software. Nucleostemin and Ki67 were not normally distributed according to the χ2-test for goodness of fit. Therefore, the Kruskal–Wallis test and Bonferroni–Dunn test were used to evaluate differences between NS and Ki67 LIs in every group. Scatter plots were used to observe the associations between proteins. The relation between the protein expression of NS and Ki-67 was investigated by Pearson’s correlation coefficient test. Student’s t-test was used to examine differences in proliferative activity between the scrambled siRNA-treated cells and NS siRNA-treated cells. P-values of less than 0.05 were regarded as statistically significant.
Immunohistochemical staining of NS and Ki67 in tissue specimens. To determine the association between NS expression and oral carcinogenesis, we first investigated the expression pattern of NS in tissue specimens using immunohistochemical staining. Immunohistochemical staining of Ki67, a marker of proliferation, was carried out at the same time. Immunoreactive Ki67 and NS were localized to the cell nucleus. In all tissue specimens, there was a higher incidence of NS-positive cells than Ki67-positive cells. Nucleostemin protein expression was determined by immunohistochemical staining of paraffin-embedded specimens of various histopathological grades covering the entire spectrum of oral carcinogenesis from normal epithelia to invasive cancer. Normal tongue tissue showed the lowest LIs for Ki67 and NS. The Ki67 and NS proteins were located mainly in the parabasal compartment. In most of the normal tissue samples, there was a characteristic absence of protein expression in the parabasal layer, especially for Ki67. By contrast, NS expression extended from the basal and suprabasal compartments to the prickle cell region (Fig. 1A). To summarize, in normal lingual epithelium tissue, Ki-67 was positive for a few parabasal cells, sporadically, but for very few basal and suprabasal/prickle cells. However, the positivities of NS were relatively high compared with Ki-67 in each layer. The positivities of NS were highest in the parabasal cell layer, and NS-positive cells were observed more frequently in basal and suprabasal/prickle cell layers compared with Ki67. But NS-positive cells in the basal and suprabasal layers were relatively low compared with the parabasal layer (Fig. S1).
The LIs of Ki67 and NS tended to increase in relation to progression of the mucosal neoplasm. The LIs were significantly elevated from moderate dysplasia to TSCC compared with those in normal tissues (P < 0.01) (Fig. 1A,B).
Scatter plots showed a positive association between the LIs of Ki67 and NS. A statistically significant (P < 0.01) correlation (r = 0.67) was observed between the labeling indices (Fig. 1C).
These results suggest that the degree of NS immunostaining is related to the proliferative activity of normal and neoplastic epithelial cells in tissue specimens.
Nucleostemin mRNA and protein expression in OSCC cell lines. We next examined the mRNA and protein of NS in cultured OSCC cell lines. For this purpose, HSC-2, HSC-3, SAS, KB, and Ca9-22 cells were used. All five cell lines contained NS mRNA, as determined using RT-PCR (Fig. 2A). Consistent with the mRNA, the protein was detected in all these cell lines with the use of Western blotting (Fig. 2B). The NS protein was also clearly detectable by immunofluorescence staining in all OSCC cell lines (Fig. 2C). The results indicated that, as in other human cell lines reported previously, the gene and protein were expressed in OSCC tumor-derived cells.
Effect of NS siRNA on cell proliferation and expression of cell cycle regulatory molecules in OSCC cell lines. To elucidate the role of NS in cell proliferation, we carried out Western blot analysis for a mitosis-associated marker. It was first proposed that the proliferation-promoting function of NS is due to its p53 binding and possibly inactivating property,(8) and for RNAi knockdown experiments, we examined changes in cell cycle regulatory molecules in both the SAS cell line, harboring wild-type TP53, and Ca9-22, harboring mutant TP53. Western blot analysis showed that levels of both NS protein and phospho histone H3 (p-H3), a mitosis-associated marker, were significantly reduced in SAS cells following NS siRNA treatment (Fig. 3A). Immunofluorescence also showed a significant decrease in NS protein in NS siRNA-treated SAS cells (Fig. 3B). Moreover, the effect of the NS knockdown on cell proliferation was evaluated using the MTT assay. There was a significant reduction in proliferation among NS siRNA-treated SAS cells compared to the cells treated with scrambled siRNA (Fig. 3C, P < 0.05). From these results, we conclude that NS plays an important role in the proliferation of cultured OSCC cells.
According to the results of Western blotting (Fig. 3D), 48 h after the transfection of OSCC cell lines with NS siRNA, the level of expression of cleaved caspase-3 (C-casp3), a marker of apoptosis, increased. Additionally, we tried to monitor changes in the expression of cell cycle regulatory molecules such as p53, p21, and p27. As shown in Figure 3(D,E), we were unable to detect any considerable changes in the expression of p53 by either Western blotting or RT-PCR. However, the expression of p21 and p27 was upregulated at both the mRNA and protein levels, in NS siRNA-treated cells compared to scrambled siRNA-treated cells. Similar results were observed in both SAS and Ca9-22 cells. These results indicate that the cell cycle arrest and increased apoptosis seen in NS knockdown cells are not dependent on p53, and that p21 and p27 were involved in the decreased OSCC cell proliferation induced by low levels of NS.
Cornification in vitro and expression pattern of NS and epithelial differentiation markers in SAS cells. To investigate whether the expression of NS changes on differentiation of the cultured SAS cells, we carried out cornification in vitro and examined the expression of NS and epithelial differentiation markers, involucrin (Invo) and cytokeratin19 (CK19). Invo is a useful marker of differentiating epithelial cells, it is expressed from the upper spinous layer, in the skin and oral mucosa including tongue.(31–33) It has also been used as a differentiation marker for OSCC, including tongue.(34–36) CK19 is a marker of the basal layer of stratified epithelia.(37) Ca2+ is a known regulator of epithelial differentiation. The increase in the intracellular Ca2+ level stimulates the terminal differentiation of OSCC cell lines in culture. The inability of OSCC cell lines to differentiate is related to a defect in achieving adequate levels of Ca2+.(38–40) Based on these reports, we regulated extracellular Ca2+ concentration by using Ca2+-free KSFM. Cornification of SAS cells was observed at a high concentration (0.5 mM) but not low concentration (0.05 mM) of extracellular Ca2+ by using rhodanile blue staining (Fig. 4A). We next compared the protein levels of NS and epithelial differentiation markers between undifferentiated SAS cells and differentiated SAS cells. According to the results of Western blot analysis (Fig. 4B), NS was expressed at similar levels in both groups. However, the expressions of Invo and CK19 differed significantly. From these results, we propose that the expression level of NS is not affected by the differentiation status of OSCC cell lines in vitro.
In the present study, we examined the possible involvement of the nucleolar protein NS in tongue carcinogenesis by comparing the NS protein expression profiles of surgically resected samples containing normal, precancerous, and cancerous tissues with those of OSCC cell lines in which endogenous NS expression was knocked down.
The first part of the study revealed that NS was highly expressed in the neoplastic tissues compared with the normal controls. Importantly, increased NS expression was also observed in the preneoplastic mucosal tissues, including mild, moderate, or severe epithelial dysplasia. This finding indicates that increased NS expression occurs very early during oral carcinogenesis and may be used as a biomarker of preneoplastic tongue lesions. Additionally, previous studies show that NS is necessary for not only ribosome biosynthesis, which is essential for cell cycle progression, but also protein synthesis in differentiation cells.(41–44) Therefore, we speculate that stem cells, cancer cells, or normal differentiated cells may all acquire NS expression when in an actively proliferating state in tongue tissues. We suppose that NS is more like a proliferation marker than a protein simply involved in the cell cycle control of stem and cancer cells. This proposal provides a reasonable explanation for what has been seen in normal tongue tissue.
We next investigated whether NS plays a functional role in the proliferation of OSCC cell lines; we chose SAS and Ca9-22 cells with similar expression of NS. Successful suppression of NS in both lines, as confirmed by Western blotting, led to a significant decrease in the number of viable cells, indicative of an important regulatory role for NS in maintaining the proliferation of these cells. The decline in the population of proliferating cells after NS suppression is consistent with previous reports.(8,14,15,19,22)
Invo is a cytoplasmic protein. It is a precursor of the epidermal cornified envelope that becomes cross-linked to increasingly insoluble, high-molecular-weight complexes during the envelope’s assembly as part of the terminal differentiation process and, as such, a useful marker for this process in the stratified squamous epithelium.(45,46) This molecule had been used as a differentiating marker for TSCC in previous studies.(34–36) CK19 is a marker of simple epithelia and of the basal layer of stratified epithelia and its levels have been reported to decrease when keratinocytes begin to produce involucrin.(37) Although NS expression was constant in the cornification assay, the cultured cells expressing high involucrin showed low CK19 levels, similar to culture derived from human oral non-keratinizing epithelia.(47) These results indicate that NS is not correlated with the expression of differentiation markers in OSCC. Our findings suggest that stem cells, cancer cells, or normal differentiated cells, may all acquire NS expression in an actively proliferating state. A recent study of skeletal muscle cell differentiation supports our findings.(44) Therefore, NS is more like a proliferation-related factor rather than a differentiation-related factor in OSCC.
The knockdown of NS led to inhibition of cellular replication and induced apoptosis in OSCC cells. It was first proposed that the proliferation-promoting function of NS is due to its p53-binding and possibly inactivating property.(48) Nevertheless, to date no experimental evidence has been presented to support the hypothesis, beyond the observation that these two proteins co-immunoprecipitate and can bind to each other, as indicated by the GST pull-down assay.(8) Thus, we tried for the first time to experimentally test the hypothesis by using two OSCC cell lines, SAS and Ca9-22, with different TP53 status. To this aim, we studied changes in the expression of p21 (also known as p21WAF1/CIP1), one of the most prominent effectors of p53, and its cell cycle regulatory function,(49) and another cell cycle regulatory molecule p27 (also known as p27KIP1). It was anticipated that downregulation of NS expression would result in a dramatic increase in expression of p21 (as its transcription is under the tight control of p53) and the activated p53 (due to absence of NS) would promote its transcription in order to repress cell cycle progression.(50) Nevertheless, our analysis revealed that downregulation of NS expression did not have any detectable impact on the expression of p21, despite an apparent reduction in cell proliferation and increase in apoptosis. This reveals that the proliferation-promoting function of NS might not be merely due to its p53-binding activity. Furthermore, similar experimentation has previously shown that downregulation of NS expression decreased the rate of propagation of p53-null HeLa cells.(19) These observations further support our claim that NS acts through mediators other than the p53 protein. In contrast, p27 expression was remarkably upregulated in both SAS and Ca9-22 cells under low-NS conditions in the siRNA experiment. p27Kip1 is associated predominantly with cyclin D–CDK4, but shows the ability to inhibit a variety of cyclin–cyclin-dependent kinase complexes in vitro.(51–53) These observations suggest that NS could regulate p27 expression and induce cell cycle arrest through this molecule.
p21 and p27 are key molecules in cell cycle progression.(51–56) In OSCC, the expression of these molecules is related to malignant phenotype and prognosis.(57–59) To elucidate the regulatory mechanism of these molecules is useful for understanding malignant behavior and controlling cancer. The present studies indicate that NS could control cell cycle arrest through the p53 independent pathway. Concomitantly, our results suggested that NS could be involved in transcriptional regulation of p27 and suppression of malignant phenotype in OSCC cell lines. However, further studies are required to elucidate its mechanism.
In previous studies, NS has been reported to function in stem cells in several tissues.(12,20,60–62) However, NS was constantly expressed in normal and neoplastic oral epithelial cells and in cultured OSCC cell lines, which does not support the notion that NS is specific to stem cells. Recently, surface markers such as CD44, CD133, and polycomb proteins have been reported to be markers of stemness for OSCC.(63,64) With the advancement and accumulation of stem cell studies in oral cancer research, the significance of NS expression in normal and neoplastic oral epithelia should become clearer.
We thank Mrs Motoko Kagayama, Mrs Takako Maeda, and Mrs Hiroko Kouzuma for their skilful technical assistance.
The authors have no conflict of interest to declare.