研究内容

The NOTCH family of membranous receptors plays key roles during development and carcinogenesis. Since NOTCH2, yet not NOTCH1 has been shown essential for murine hepatogenesis, NOTCH2 rather than NOTCH1 may be more relevant to human hepatocarcinogenesis; however, no previous studies have supported this hypothesis. We therefore assessed the role of NOTCH2 in human hepatocellular carcinoma (HCC) by immunohistochemistry and cell culture. Immunohistochemically, 19% of primary HCCs showed nuclear staining for NOTCH2, indicating activated NOTCH2 signaling. NOTCH2-positive HCCs were on average in more advanced clinical stages, and exhibited more immature cellular morphology, i.e. higher nuclear-cytoplasmic ratios and nuclear densities. Such features were not evident in NOTCH1 positive HCCs. In human HCC cell lines, abundant NOTCH2 expression was associated with anaplasia, represented by loss of E-cadherin. When NOTCH2 signaling was stably downregulated in HLF cells, an anaplastic HCC cell line, the cells were attenuated in potential for in vitro invasiveness and migration, as well as in vivo tumorigenicity accompanied by histological maturation. Generally, inverse results were obtained for a differentiated HCC cell line, Huh7, manipulated to overexpress activated NOTCH2. These findings suggested that the NOTCH2 signaling may confer aggressive behavior and immature morphology in human HCC cells.

Expression of fascin-1, an actin bundling protein, is a poor prognostic factor in hepatocellular carcinoma (HCC). However, its biological role in HCC cells remains unclear. Using human HCC tissues and cell lines HLE, Hep3B, and Huh7, we investigated whether fascin-1 is involved in epithelial-mesenchymal transition (EMT) and increases invasiveness, thus serving as a promoter of cancer aggressiveness. Immunohistochemical analysis revealed that fascin-1 expression in 19% of primary HCCs was associated with repression of E-cadherin expression, indicating EMT. In vitro, HLE cells showed high fascin-1 expression, loss of E-cadherin, and efficient invasion through Matrigel. Knockdown of fascin-1 significantly repressed invasiveness of the HLE cells and slightly induced E-cadherin expression. In contrast, Huh7 cells had low fascin-1 levels, high E-cadherin expression, and were expectedly non-invasive. However, forced overexpression of fascin-1 conferred only modest invasiveness without E-cadherin repression, indicating that fascin-1 alone cannot effectively stimulate invasiveness or EMT. Furthermore, Hep3B cells were non-invasive despite high fascin-1 expression. Nevertheless, fascin-1 overexpression dramatically increased the migratory potential of Huh7 cells. We then evaluated matrix metalloproteinases (MMPs) 2 and 9 from the HCC cell lines. Significant MMP secretion was only found in HLE cells. Although MMP levels were not elevated in fascin-1-overexpressing Huh7 cells, their invasiveness was remarkably augmented by coculture with HLE cells, and was suppressed in the presence of an MMP inhibitor. In conclusion, we propose that fascin-1 primarily acts as a migration factor associated with EMT in HCC cells and facilitates their invasiveness in combination with MMPs.

For melanoma treatment, an early diagnosis and a complete resection of the primary tumor is essential. In addition, detection of factors that may be related to metastasis is indispensable. A total of 30 Japanese patients with Stage I or II melanoma, diagnosed according to the classification of the American Joint Committee on Cancer, are included in this study. Clinical background (sex, onset age, primary tumor area, existence of remaining cancer cells at the resected tissue margin, and treatment after the primary surgery) and immunohistochemical staining (Nestin and Fascin) on the resected tissue were examined to detect factors statistically related to metastasis. The analysis result has shown that older onset age and positive immunohistochemical expressions of Nestin and Fascin are statistically related to metastasis. To facilitate meticulous observation of Nestin and Fascin expression at different timing (e.g., onset and metastasis), double immunofluorescence staining was performed. Nestin is a class VI intermediate filament protein, initially detected in neural stem cells. Fascin is an actin-bundling protein which regulates cell adhesion, migration and invasion. Nestin and Fascin are suggested to relate to melanoma metastasis, however, the potential role of Fascin is controversial. Analysis of variations in Fascin expression detected in this study may contribute to further investigations concerning potential roles of Fascin for progression of melanoma. This is the first study to report double immunofluorescent staining of Nestin and Fascin in melanoma. Nestin and Fascin double-positive melanoma cells were detected.

Liver cancer is one of the most prevalent cancers in Japan with hepatocellular carcinoma (HCC) as the major histological subtype. Successful novel treatments for HCC have been reported; however, recurrences or metastasis may occur, which results in poor prognoses and high mortality of HCC patients. Fascin, an actin-bundling protein, regulates cell adhesion, migration, and invasion. Its overexpression positively correlates with poor prognosis of malignant tumors, and Fascin is considered as one of the tumor biomarkers and therapeutic target proteins. In this study, we attempted to reveal the relationship between Fascin and HCC using HLE, one of the human HCC cell lines. We performed the study with classical immunocytochemistry and recently developed techniques, such as wound-healing assay, spheroid cultivation, and low-vacuum scanning electron microscopy (LV-SEM). Non-Fascin-knockdown (FKD) cell spheroid had a regular spherical appearance with tight cell-cell connections, while FKD cell spheroid had an irregular shape with loose cell-cell connections. Cells of non-FKD spheroid presented fibrous protrusions on the cell surface, contrarily, cells of FKD spheroids showed bulbous-shaped protrusions. Morphological observation of FKD and non-FKD HLE spheroids were performed using LV-SEM. Our study may help to reveal the roles of Fascin in the process of HCC formation and its malignancy.