Shojiro Kitajima

Invasion and Metastasis of Oral Cancer Cells Require Methylation of E-Cadherin and/or Degradation of Membranous β-Catenin

The extent of lymph node metastasis is a major determinant in the prognosis of oral squamous cell carcinoma (OSCC). Abnormalities of cell adhesion molecules are known to play an important role in invasion and metastasis of cancer cells through the loss of cell-to-cell adhesion. In this study, we isolated highly invasive clones from an OSCC cell line established from a lymph node metastasis by using an in vitro invasion assay method and compared the abnormalities of cell adhesion molecule E-cadherin and β-catenin in these cells. The isolated, highly invasive clones showed significant invasive capacity and reduction of E-cadherin and membranous β-catenin protein in comparison with parent cells. We found that reduced expression of E-cadherin was due to methylation of its promoter region. In fact, most invasive and metastatic area of OSCCs showed reduced expression and methylation of E-cadherin. Moreover, we found that reduced expression of membranous β-catenin was due to its protein degradation. Reduced expression of membranous β-catenin was also found frequently in invasive and metastatic areas of OSCCs. In summary, invasion and metastasis of OSCC cells require methylation of E-cadherin and/or degradation of membranous β-catenin. In addition, we suggest that the method of isolation of highly invasive clones may be useful for studies aimed at discovering novel genes involved in invasion and metastasis.

Periostin Promotes Invasion and Anchorage-Independent Growth in the Metastatic Process of Head and Neck Cancer

Head and neck squamous cell carcinoma (HNSCC) is one of the most common types of human cancer. Typically, HNSCC cells show persistent invasion that frequently leads to local recurrence and distant lymphatic metastasis. However, molecular mechanisms associated with the invasion and metastasis of HNSCC remain poorly understood. Here, we identified periostin as an invasion-promoting factor in HNSCC by comparing the gene expression profiles between parent HNSCC cells and a highly invasive clone. Indeed, periostin overexpression promoted invasion and anchorage-independent growth both in vitro and in vivo in HNSCC cells. Moreover, periostin-overexpressing cells spontaneously metastasized to cervical lymph nodes and to the lung through their aggressive invasiveness in an orthotopic mouse model of HNSCC. Interestingly, periostin was highly expressed in HNSCCs in comparison with normal tissues, and the level of periostin expression was well correlated with the invasiveness of HNSCC cases. In summary, these findings suggest that periostin plays an important role in the invasion and anchorage-independent growth of HNSCC.

Periostin is frequently overexpressed and enhances invasion and angiogenesis in oral cancer

Oral squamous-cell carcinoma (OSCC) is one of the most common types of human cancer. Typically OSCC cells show persistent invasion that frequently leads to local recurrence and distant lymphatic metastasis. We previously identified Periostin as the gene demonstrating the highest fold change expression in the invasive clone by comparing the transcriptional profile of parent OSCC cell line and a highly invasive clone. Here, we demonstrated that Periostin overexpression enhanced invasiveness in oral cancer cell lines. To know the role of Periostin in invasion, angiogenesis and metastasis in OSCC cases, we first examined the expression of Periostin mRNA in 31 OSCC cases by RT–PCR and Periostin protein in 74 OSCC cases by immunohistochemistry. Then, we compared the Periostin expression with invasion pattern, metastasis and blood vessel density. Periostin mRNA and protein overexpression were frequently found in OSCC cases and Periostin expression was well correlated with the invasion pattern and metastasis. Moreover, blood vessel density of Periostin-positive cases was higher than those of Periostin-negative cases. Interestingly, recombinant Periostin enhanced capillary formation in vitro in a concentration-dependant manner. In summary, these findings suggest that Periostin may promote invasion and angiogenesis in OSCC, and that Periostin can be a strong marker for prediction of metastasis in oral cancer patients.

Overexpression of Aurora-A potentiates HRAS-mediated oncogenic transformation and is implicated in oral carcinogenesis.

Aurora kinases are known to play a key role in maintaining mitotic fidelity, and overexpression of aurora kinases has been noted in various tumors. Overexpression of aurora kinase activity is thought to promote cancer development through a loss of centrosome or chromosome number integrity. Here we observed augmentation of G12V-mutated HRAS-induced neoplastic transformation in BALB/c 3T3 A31-1-1 cells transfected with Aurora-A. Aurora-A-short hairpin RNA (shRNA) experiments showed that the expression level of Aurora-A determines susceptibility to transformation. Aurora-A gene amplification was noted in human patients with tongue or gingival squamous carcinoma (4/11). Amplification was observed even in pathologically normal epithelial tissue taken at sites distant from the tumors in two patients with tongue cancer. However, overexpression of Aurora-A mRNA was observed only within the tumors of all patients examined (11/11). Our data indicate that Aurora-A gene amplification and overexpression play a role in human carcinogenesis, largely due to the effect of Aurora-A on oncogenic cell growth, rather than a loss of maintenance of centrosomal or chromosomal integrity.

Aurora-B/AIM-1 kinase activity is involved in Ras-mediated cell transformation

Aurora-B, previously known as AIM-1, is a conserved eukaryotic mitotic protein kinase. In mammals, this kinase plays an essential role in chromosomal segregation processes, including chromosome condensation, alignment, control of spindle checkpoints, chromosome segregation, and cytokinesis. Aurora-B is overexpressed in various cancer cells, suggesting that the kinase activity perturbs chromosomal segregation processes. Its forced overexpression induces chromosomal number instability and progressive tumorigenicity in rodent cells in vitro and in vivo. Nevertheless, based on focus formation in BALB/c 3T3 A31-1-1 cells, Aurora-B is not oncogenic. Here, we show that Aurora-B kinase activity augments Ras-mediated cell transformation. RNA interference with short hairpin RNA inhibits transformation by Ras and its upstream oncogene Src, but not by the downstream oncogene Raf. In addition, the inner centromere protein, which is a passenger protein associated with Aurora-B, has a similar ability to potentiate the activity of oncogenic Ras. These data indicate that elevated Aurora-B activity promotes transformation by oncogenic Ras by enhancing oncogenic signaling and by converting chromosome number-stable cells to aneuploid cells.

Role of Cks1 overexpression in oral squamous cell carcinomas: cooperation with Skp2 in promoting p27 degradation.

Down-regulation of p27 is frequently observed in various cancers due to an enhancement of its degradation. Skp2 is required for the ubiquitination and consequent degradation of p27 protein. Another protein called Cks1 is also required for p27 ubiquitination in the SCF(Skp2) ubiquitinating machinery. In the present study, we examined Cks1 expression and its correlation with p27 in oral squamous cell carcinoma (OSCC) derived from tongue and gingiva. By immunohistochemical analysis, high expression of Cks1 was present in 62% of OSCCs in comparison with 0% of normal mucosae. In addition, 65% of samples with low p27 expression displayed high Cks1 levels. Finally, Cks1 expression was well correlated with Skp2 expression and poor prognosis. To study the role of Cks1 overexpression in p27 down-regulation, we transfected Cks1 with or without Skp2 into OSCC cells. Cks1 transfection could not induce a p27 down-regulation by itself, but both Cks1 and Skp2 transfection strongly induced. Moreover, we inhibited Cks1 expression by small interference RNA (siRNA) in OSCC. Cks1 siRNA transfection induced p27 accumulation and inhibited the growth of OSCC cells. These findings suggest that Cks1 overexpression may play an important role for OSCC development through Skp2-mediated p27 degradation, and that Cks1 siRNA can be a novel modality of gene therapy.

Small interfering RNA targeting of S phase kinase-interacting protein 2 inhibits cell growth of oral cancer cells by inhibiting p27 degradation.

S phase kinase–interacting protein 2 (Skp2), an F box protein, is required for the ubiquitination and consequent degradation of p27. It is well known that reduced expression of p27 is frequently observed in various cancers including oral squamous cell carcinoma and is due to an enhancement of its protein degradation. Our previous study showed that overexpression of Skp2 was frequently found in oral squamous cell carcinoma and inversely correlated with p27 expression. Recently, a technique known as RNA interference has been successfully adapted to mammalian cells. In the present study, we investigated if small interfering RNA (siRNA)-mediated gene silencing of Skp2 can be employed in order to inhibit p27 down-regulation in oral squamous cell carcinoma. We used a siRNA plasmid vector, which has an advantage over synthetic siRNAs in determining the effects of decreasing the high constitutive levels of Skp2 protein in oral squamous cell carcinoma. We showed that Skp2 siRNA transfection decreased Skp2 protein and induced the accumulation of p27 protein in oral squamous cell carcinoma cells. Moreover, p27 protein in Skp2 siRNA-transfected cells is more stabilized than that in control siRNA-transfected cells. Interestingly, Skp2 siRNA inhibited the cell proliferation of oral squamous cell carcinoma cells both in vitro and in vivo. Our findings suggest that siRNA-mediated gene silencing of Skp2 can be a novel modality of cancer gene therapy for suppression of p27 down-regulation.

Constitutive Phosphorylation of Aurora-A on Ser51 Induces Its Stabilization and Consequent Overexpression in Cancer

Background The serine/threonine kinase Aurora-A (Aur-A) is a proto-oncoprotein overexpressed in a wide range of human cancers. Overexpression of Aur-A is thought to be caused by gene amplification or mRNA overexpression. However, recent evidence revealed that the discrepancies between amplification of Aur-A and overexpression rates of Aur-A mRNA were observed in breast cancer, gastric cancer, hepatocellular carcinoma, and ovarian cancer. We found that aggressive head and neck cancers exhibited overexpression and stabilization of Aur-A protein without gene amplification or mRNA overexpression. Here we tested the hypothesis that aberration of the protein destruction system induces accumulation and consequently overexpression of Aur-A in cancer. Principal Findings Aur-A protein was ubiquitinylated by APCCdh1 and consequently degraded when cells exited mitosis, and phosphorylation of Aur-A on Ser51 was observed during mitosis. Phosphorylation of Aur-A on Ser51 inhibited its APCCdh1-mediated ubiquitylation and consequent degradation. Interestingly, constitutive phosphorylation on Ser51 was observed in head and neck cancer cells with protein overexpression and stabilization. Indeed, phosphorylation on Ser51 was observed in head and neck cancer tissues with Aur-A protein overexpression. Moreover, an Aur-A Ser51 phospho-mimetic mutant displayed stabilization of protein during cell cycle progression and enhanced ability to cell transformation. Conclusions/Significance Broadly, this study identifies a new mode of Aur-A overexpression in cancer through phosphorylation-dependent inhibition of its proteolysis in addition to gene amplification and mRNA overexpression. We suggest that the inhibition of Aur-A phosphorylation can represent a novel way to decrease Aur-A levels in cancer therapy.

Transfection of p27Kip1 Threonine Residue 187 Mutant Type Gene, Which Is Not Influenced by Ubiquitin-Mediated Degradation, Induces Cell Cycle Arrest in Oral Squamous Cell Carcinoma Cells

Objective: It is well known that reduction of the cyclin-dependent kinase inhibitor p27Kip1 protein correlates with the malignant behavior of various cancers including oral squamous cell carcinoma (OSCC). The loss of p27Kip1 protein is suggested to be due to the enhancement of its posttranslational degradation. In the present study, to evaluate the effects of p27Kip1 transfection on the cell cycle, we transfected OSCC cell lines with a high activity of p27Kip1 degradation with p27Kip1 threonine 187-to-alanine (T187A) mutant gene, which is not influenced by ubiquitin-mediated degradation, as well as with wild type gene. Methods: We transfected p27Kip1 T187A mutant and wild type gene into OSCC cell lines (HSC2 and HSC3) by using an ecdysone-inducible gene expression system. Results: After treatment with ponasterone A, we could find an induction of both p27Kip1 wild type and T187A mutant protein. Both wild type and T187A mutant protein induced by 5 µM ponasterone A inhibited cell growth and increased cell number at the G1 phase. After treatment with 1 µM ponasterone A, ectopic p27Kip1 protein was degraded in wild type clones, but not in T187A mutant clones. Moreover, transfection of the T187A mutant gene was more effective in inhibiting cell growth even by induction of a small amount of protein. Conclusion: We suggest that the transfection of the p27Kip1 T187A mutant gene can be a modality of cancer gene therapy for OSCC.

Oncogenic role of nuclear accumulated Aurora-A

Aurora‐A, also known as Aik, BTAK, or STK15, is a centrosomal serine/threonine protein kinase, which is proto‐oncogenic and is overexpressed in a wide range of human cancers. Besides gene amplification and mRNA overexpression, proteolytic resistance mechanisms are thought to contribute to overexpression of Aurora‐A. However, it is not yet clear how overexpressed Aurora‐A affects the expression of transformed phenotype. Here, we found that nuclear accumulation of Aurora‐A was critical for transformation activity. Cellular protein fractionation experiments and immunoblot analysis demonstrated a predominance of Aurora‐A in the nuclear soluble fraction in head and neck cancer cells. Indirect immunofluorescence using confocal laser microscopy confirmed nuclear Aurora‐A in head and neck cancer cells, while most oral keratinocytes exhibited only centrosomal localization. The expression of nuclear export signal‐fused Aurora‐A demonstrated that the oncogenic transformation activity was lost on disruption of the nuclear localization. Thus, the cytoplasmic localization of overexpressed Aurora‐A previously demonstrated by immunohistochemical analysis is not likely to correspond to that in intact cancer cells. This study identifies an alternative mode of Aurora‐A overexpression in cancer, through nuclear rather than cytoplasmic functions. We suggest that substrates of Aurora‐A in the cell nuclear soluble fraction can represent a novel therapeutic target for cancer.