Takanori Tsuji

Epithelial-Mesenchymal Transition and Cell Cooperativity in Metastasis

The role of epithelial-mesenchymal transition (EMT) in metastasis remains controversial. EMT has been postulated as an absolute requirement for tumor invasion and metastasis. Three different models including incomplete EMT, mesenchymal-epithelial transition (MET), and collective migration have been proposed for the role of EMT in cancer invasion and metastasis. However, skepticism remains about whether EMT truly occurs during cancer progression, and if it does, whether it plays an indispensible role in metastasis. Our recent findings suggest that EMT cells are responsible for degrading the surrounding matrix to enable invasion and intravasation of both EMT and non-EMT cells. Only non-EMT cells that have entered the blood stream are able to re-establish colonies in the secondary sites. Here, we discuss an alternative model for the role of EMT in cancer metastasis in which EMT and non-EMT cells cooperate to complete the entire process of spontaneous metastasis.

Endogenous angiogenin in endothelial cells is a general requirement for cell proliferation and angiogenesis.

Angiogenin is an angiogenic protein that undergoes nuclear translocation in endothelial cells where it accumulates in the nucleolus and stimulates rRNA transcription, a rate-limiting step in ribosome biogenesis, protein translation, and cell growth. Here, we report that angiogenin is required for cell proliferation induced by various other angiogenic proteins including acidic and basic fibroblast growth factors (aFGF and bFGF), epidermal growth factor (EGF), and vascular endothelial growth factor (VEGF). Downregulation of angiogenin in endothelial cells by small interfering RNA (siRNA) and antisense results in a decrease in rRNA transcription, ribosome biogenesis, and cell proliferation induced by these angiogenic factors. Inhibitors of the nuclear translocation of angiogenin abolish the angiogenic activities of these factors. Stable angiogenin antisense transfection in HeLa cells reduces tumor angiogenesis in athymic mice despite the elevated expression level of bFGF and VEGF. Thus, nuclear angiogenin assumes an essential role in endothelial cell proliferation and is necessary for angiogenesis induced by other angiogenic factors. Angiogenin-stimulated rRNA transcription in endothelial cells may thus serve as a crossroad in the process of angiogenesis induced by various angiogenic factors.

Epithelial-mesenchymal transition induced by growth suppressor p12CDK2-AP1 promotes tumor cell local invasion but suppresses distant colony growth

Epithelial-mesenchymal transition (EMT) has been considered essential for metastasis, a multistep process including local invasion, intravasation, extravasation, and proliferation at distant sites. However, controversy remains as to whether EMT truly happens and how important it is to metastasis. We studied the involvement of EMT in individual steps of metastasis and found that p12(CDK2-AP1), a down-stream effector of transforming growth factor beta, induced EMT of hamster cheek pouch carcinoma-1 cells by promoting the expression of Twist2. EMT cells have an increased invasive but decreased metastatic phenotype. When s.c. inoculated, both EMT and non-EMT cells established primary tumors, but only EMT cells invaded into the adjacent tissues and blood vessels; however, neither cells formed lung metastases. When i.v. inoculated, only non-EMT cells established lung metastases. Moreover, s.c. inoculation of a mixture of the two cell types resulted in intravasation of both cell types and formation of lung metastasis from non-EMT cells. Our results allowed us to propose a novel model for the role of EMT in cancer metastasis. We showed that EMT and non-EMT cells cooperate to complete the spontaneous metastasis process. We thus hypothesize that EMT cells are responsible for degrading the surrounding matrix to lead the way of invasion and intravasation. Non-EMT cells then enter the blood stream and reestablish colonies in the secondary sites.

Angiogenin is translocated to the nucleus of HeLa cells and is involved in ribosomal RNA transcription and cell proliferation

Angiogenin is an angiogenic protein known to play a role in rRNA transcription in endothelial cells. Nuclear translocation of angiogenin in endothelial cells decreases as cell density increases and ceases when cells are confluent. Here we report that angiogenin is constantly translocated to the nucleus of HeLa cells in a cell density-independent manner. Down-regulation of angiogenin expression by antisense and RNA interference results in a decrease in rRNA transcription, ribosome biogenesis, proliferation, and tumorigenesis both in vitro and in vivo. Exogenous angiogenin rescues the cells from antisense and RNA interference inhibition. The results showed that angiogenin is constitutively translocated into the nucleus of HeLa cells where it stimulates rRNA transcription. Thus, besides its angiogenic activity, angiogenin also plays a role in cancer cell proliferation.

A therapeutic target for prostate cancer based on angiogenin-stimulated angiogenesis and cancer cell proliferation

Human angiogenin is progressively up-regulated in the prostate epithelial cells during the development of prostate cancer from prostate intraepithelial neoplasia (PIN) to invasive adenocarcinoma. Mouse angiogenin is the most up-regulated gene in AKT-induced PIN in prostate-restricted AKT transgenic mice. These results prompted us to study the role that angiogenin plays in prostate cancer. Here, we report that, in addition to its well established role in mediating angiogenesis, angiogenin also directly stimulates prostate cancer cell proliferation. Angiogenin undergoes nuclear translocation in PC-3 human prostate cancer cells grown both in vitro and in mice. Thus, knocking down angiogenin expression in PC-3 human prostate adenocarcinoma cells inhibits ribosomal RNA transcription, in vitro cell proliferation, colony formation in soft agar, and xenograft growth in athymic mice. Blockade of nuclear translocation of angiogenin by the aminoglycoside antibiotic neomycin inhibited PC-3 cell tumor growth in athymic mice and was accompanied by a decrease in both cancer cell proliferation and angiogenesis. These results suggest that angiogenin has a dual effect, angiogenesis and cancer cell proliferation, in prostate cancer and may serve as a molecular target for drug development. Blocking nuclear translocation of angiogenin could have a combined benefit of antiangiogenesis and chemotherapy in treating prostate cancer.

The nuclear function of angiogenin in endothelial cells is related to rRNA production.

Angiogenin is a potent angiogenic protein whose inhibition is known to prevent human tumor growth in athymic mice. It is secreted by both tumor and normal cells; and interacts with endothelial and smooth muscle cells to induce a wide range of cellular responses including cell migration and invasion, proliferation, and formation of tubular structures. Angiogenin is rapidly endocytosed and translocated to the cell nucleus where it accumulates in the nucleolus and binds to DNA. Although nuclear translocation is necessary for its angiogenic activity, the nuclear function of angiogenin is unclear. Here we report that exogenous angiogenin enhances the production of 45S rRNA in endothelial cells, and reduction of endogenous angiogenin inhibits its transcription. In a nuclear run-on assay, angiogenin stimulates RNA synthesis including that containing the initiation site sequences of 45S rRNA. This suggests that the nuclear function of angiogenin relates to its capacity to induce rRNA synthesis. Because rRNA transcription is essential for the synthesis of new ribosomes that are necessary for protein translation and cell growth, inhibition of angiogenin-stimulated transcription of rRNA may inhibit angiogenesis and therefore, would serve as a molecular target for therapeutic intervention.

Deleted in oral cancer-1 (doc-1), a novel oral tumor suppressor gene.

We have identified, isolated, and par‐tially characterized doc‐1, a novel cDNA sequence whose activity is consistent with a suppressor of hamster oral carcinogenesis. Doc‐1 is an evolutionarily conserved gene exhibiting loss of heterozygosity and marked reduction in expression in malignant hamster oral keratinocytes. The full‐length doc‐1 cDNA encodes an 87 amino acid product that shows a significant homology to one of the seven novel genes induced in mouse fibroblasts by TNF‐α. Transfection of the full‐length doc‐1 cDNA into malignant hamster oral keratinocytes alters the behavior of the recipients in terms of morphology, growth rate, and anchorage‐independent growth, suggesting reversion of transformation phenotypes. We propose that doc‐1 is a novel tumor suppressor gene in oral cancer development.—Todd, R., McBride, J., Tsuji, T., Donoff, R. B., Nagai, M., Chou, M. Y., Chiang, T., Wong, D. T. W. Deleted in oral cancer‐1 (doc‐1), a novel oral tumor suppressor gene. FASEB J. 9, 1362‐1370(1995)

The E-cadherin gene is silenced by CpG methylation in human oral squamous cell carcinomas

Reduction of E‐cadherin strongly relates to invasiveness and metastasis in vitro. To clarify CpG methylation around the promoter region of the E‐cadherin gene in oral squamous cell carcinoma (SCC), we examined the DNA samples of various human SCC cell lines and primary oral SCC tissues by methylation‐specific polymerase chain reaction (MSP). CpG methylation of the E‐cadherin gene markedly correlated to the reduction of E‐cadherin expression in human oral SCC cell lines. In primary oral SCC tissues, only 1 of 5 preserved E‐cadherin‐expressing tissues was methylated, whereas methylation was found in 17 (94.4%) of 18 E‐cadherin‐reduced tissues. Our results suggest that reduction of E‐cadherin expression is associated with CpG methylation of the E‐cadherin gene promoter. We recently established two cell lines with high and low metastatic potential, UM1 and UM2, from SCC primary tongue tissue of a patient. E‐cadherin expression of high‐metastatic UM1 was clearly lower than that of low‐metastatic UM2, and MSP results showed CpG methylation in the UM1 but not the UM2 cell line. To investigate whether demethylation of CpG methylation of the E‐cadherin gene could restore expression and function of E‐cadherin, we treated UM1 with the demethylating agent 5‐azacytidine (5‐aza) and found that E‐cadherin expression was indeed restored by demethylation. Moreover, in the demethylated UM1, invasion of the collagen gel was clearly suppressed compared with the untreated UM1. These results suggested that inactivation of E‐cadherin expression resulted from CpG methylation of the gene promoter; a correlation between CpG methylation of the E‐cadherin gene promoter and invasive potential was also suggested.

p12(DOC-1) is a novel cyclin-dependent kinase 2-associated protein.

ABSTRACT Regulated cyclin-dependent kinase (CDK) levels and activities are critical for the proper progression of the cell division cycle. p12DOC-1 is a growth suppressor isolated from normal keratinocytes. We report that p12DOC-1 associates with CDK2. More specifically, p12DOC-1 associates with the monomeric nonphosphorylated form of CDK2 (p33CDK2). Ectopic expression of p12DOC-1 resulted in decreased cellular CDK2 and reduced CDK2-associated kinase activities and was accompanied by a shift in the cell cycle positions of p12DOC-1transfectants (↑ G1 and ↓ S). The p12DOC-1-mediated decrease of CDK2 was prevented if the p12DOC-1 transfectants were grown in the presence of the proteosome inhibitor clasto-lactacystin β-lactone, suggesting that p12DOC-1 may target CDK2 for proteolysis. A CDK2 binding mutant was created and was found to revert p12DOC-1-mediated, CDK2-associated cell cycle phenotypes. These data support p12DOC-1 as a specific CDK2-associated protein that negatively regulates CDK2 activities by sequestering the monomeric pool of CDK2 and/or targets CDK2 for proteolysis, reducing the active pool of CDK2.

Molecular Biology of Human Oral Cancer

The application of molecular biological tools to the study of cancer has significantly advanced the field of human cancer research. Such study has demonstrated the involvement of two classes of highly conserved cellular genes in the malignant transformation process: oncogenes and tumor suppressor genes. Despite these advances in the molecular biology of human cancers, our understanding of human oral cancer lags behind that of cancer of other body sites. This review attempts to assess the current status of the molecular biology of human oral cancer.