Regulation of the actin cytoskeleton may play a crucial role in cell motility and cancer invasion. We have produced a monoclonal antibody (NCC- Lu-632, IgM, k) reactive with an antigenic protein that is upregulated upon enhanced cell movement. The cDNA for the antigen molecule was found to encode a novel isoform of nonmuscle α-actinin. This isoform (designated actinin-4) was concentrated in the cytoplasm where cells were sharply extended and in cells migrating and located at the edge of cell clusters, but was absent from focal adhesion plaques or adherens junctions, where the classic isoform (actinin-1) was concentrated. Actinin-4 shifted steadily from the cytoplasm to the nucleus upon inhibition of phosphatidylinositol 3 kinase or actin depolymerization. The cytoplasmic localization of actinin-4 was closely associated with an infiltrative histological phenotype and correlated significantly with a poorer prognosis in 61 cases of breast cancer. These findings suggest that cytoplasmic actinin-4 regulates the actin cytoskeleton and increases cellular motility and that its inactivation by transfer to the nucleus abolishes the metastatic potential of human cancers.

No abstract

We developed an integrated platform consisting of machinery and software modules that can apply vast amounts of data generated by nanoflow LC-MS to differential protein expression analyses. Unlabeled protein samples were completely digested with modified trypsin and separated by low speed (200 nl/min) one-dimensional HPLC. Mass spectra were obtained every 1 s by using the survey mode of a hybrid Q-TOF mass spectrometer and displayed in a two-dimensional plane with m/z values along the x axis, and retention time was displayed along the y axis. The time jitter of nano-LC was adjusted using newly developed software based on a dynamic programming algorithm. The comprehensiveness (60,000 -160,000 peaks above the predetermined threshold detectable in 60-g cell protein samples), reproducibility (average coefficient of variance of 0.35-0.39 and correlation coefficient of over 0.92 between duplicates), and accurate quantification with a wide dynamic range (over 10

Salivary gland tumors are relatively rare and morphologically diverse and heterogeneous tumors; therefore, histogenesis-based tumor markers are sorely needed to aid in diagnosing and determining the cell type of origin. SRY-related HMG-box 10 (SOX10) protein is a transcription factor known to be crucial in the specification of the neural crest and maintenance of Schwann cells and melanocytes. In addition, positive expression has also been implicated in the major salivary gland. Here, we examined SOX10 expression in various salivary gland tumors to correlate this expression with myoepithelial markers. Overall, 76 malignant and 14 benign tumors were examined. SOX10 expression clearly delineated two distinct subtypes of human salivary gland tumors; acinic cell carcinomas, adenoid cystic carcinomas, epithelial-myoepithelial carcinomas, myoepithelial carcinomas, and pleomorphic adenomas, including the pleomorphic adenoma component of carcinoma, were SOX10 positive, while salivary duct carcinomas, mucoepidermoid carcinomas, an oncocytic carcinoma, Oncocytomas, and Warthin tumors were SOX10 negative. Also, SOX10 was expressed in solid-type or nonspecific morphology salivary gland tumors, but was not expressed in poorly differentiated squamous cell carcinomas. In normal human salivary gland tissue, SOX10 expression was specific to the nuclei of acini and both luminal and abluminal cells of intercalated ducts but not in other sites. Moreover, the murine model suggested that SOX10 continued to be expressed from the developmental stage to adulthood in the acinar and both luminal and abluminal intercalated ducts in the major salivary gland. Thus, SOX10 is a novel marker for diagnosing and understanding the histogenesis of salivary gland tumors.

Purpose: An invasive growth pattern is one of the hallmarks of pancreatic ductal carcinoma.Actinin-4 is an actin-binding protein associated with enhanced cell motility, invasive growth, and lymph node metastasis. Actinin-4 might play an important role in the development and progression of pancreatic cancer. Experimental Design: The expression of actinin-4 was examined immunohistochemically in 173 cases of invasive pancreatic ductal carcinoma. The copy number of the actinin-4 (ACTN4) gene was calculated by fluorescence in situ hybridization. The expression of actinin-4 was stably knocked down by short hairpin RNA, and tumorigenicity was evaluated by orthotopic implantation into mice with severe combined immunodeficiency. Results: The expression level of actinin-4 was increased in 109 (63.0%) of 173 cases of pancreatic cancer. Kaplan-Meier survival curves revealed that patients with increased expression of actinin-4 had a significantly poorer outcome (P = 0.00001, log-rank test). Multivariate analysis by the Cox proportional hazard model showed that high expression of actinin-4 was the most significant independent negative predictor of survival (hazard ratio, 2.33; P = 0.000009). Amplification (defined as more than four copies per interphase nucleus) of the ACTN4 gene was detected in 11 (37.9%) of 29 cases showing increased expression of actinin-4. Knockdown of actinin-4 expression inhibited the destructive growth of cancer cells in the pancreatic parenchyma. Conclusion: Recurrent amplification of chromosome 19q13.1-2 has been reported in pancreatic cancer, but the exact target gene has not been identified. Actinin-4 contributes to the invasive growth of pancreatic ductal carcinoma, and ACTN4 is one of the candidate oncogenes in this chromosome locus.Invasive ductal carcinoma of the pancreas is one of the most aggressive forms of human malignancy, with a 5-year survival rate of <5% to 10% and a median survival of <6 months (1, 2). As a result, pancreatic cancer is the fourth leading cause of cancer death in the United States, and is the fifth in Japan (3). Massive local invasion to adjacent organs and/or metastasis to regional lymph nodes and distal organs are detected in the majority of patients at the time of diagnosis. To improve the prognosis of patients with pancreatic cancer, it will be necessary to elucidate the molecular mechanisms causing invasion and metastasis.We have identified an actin-binding protein, actinin-4, as a biomarker of cancer invasion and metastasis (4). The expression of actinin-4 was closely associated with the invasive phenotype of breast cancer and was a prognostic indicator in patients with this disease (4). A microarray analysis revealed that actinin-4 was a significant prognostic indicator in patients with non -small cell lung cancer (5). The expression level of actinin-4 protein was increased in the majority of cases of colorectal cancer, and the increase in expression was most significant in dedifferentiated cancer cells infiltrating at the invasive front (6). In mouse mo...