Manabu Iwadate

Cathepsin L is highly expressed in gastrointestinal stromal tumors

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract that are diagnosed by c-kit staining in most cases. A lysosomal cysteine proteinase termed cathepsin L has been commonly associated with malignancy in several cancer types, but this finding has not been reported for GISTs. We analyzed the cathepsin L mRNA and protein expression in GISTs. Real-time reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that cathepsin L levels were higher in GISTs than those in gastric or colorectal tumors; this finding was supported by results of the Western blot analysis. Immunohistochemistry revealed that cathepsin L was localized to the cytoplasm of GIST cells as an intense granular signal, which was not observed in the cells of leiomyoma, a mesenchymal tumor that was analyzed as a control specimen. Double immunofluorescence microscopy revealed that a portion of the granular signal colocalized with lysosome-associated membrane protein-1 (LAMP-1), which is a lysosomal marker. Moreover, immunohistochemical analysis of 43 tumor specimens revealed that 86.0% (n=37) were cathepsin-L positive, and this positivity was significantly correlated with c-kit positivity but not with other clinicopathological factors, including gender, age, region, size, mitosis and risk of recurrence. From these results, we conclude that cathepsin L is highly expressed in GISTs compared to its expression in other cancerous lesions; this identifies cathepsin-L as a new diagnostic marker for GISTs.

The role of TGF-β in digestive organ disease

More than 30 members of the TGFsuperfamily have been described in mammals, including activins and bone-morphogenetic proteins. TGFhas a dimeric structure consisting of 112 amino acids with a molecular weight of Mr 25000. There are three isoforms in humans: TGF1, TGF2, and TGF3. These isoforms are highly conserved in mammals and expressed in a tissue-specific fashion.9 The TGF1 messenger RNA (mRNA) is localized to endothelial, hematopoietic, and connective tissue cells. TGF2 mRNA is expressed in epithelial and neural cells. TGF3 is expressed in mesenchymal cells. A critical function of the TGFsignaling pathway is the inhibition of cell proliferation in hematopoietic, endothelial, epithelial, neural, and mesenchymal cells. TGFalso regulates cell differentiation, embryonic development, wound healing, and angiogenesis. TGFinitiates signaling by binding specific receptor complexes that activate Smad transcription factors. The receptor is composed of two different protein subunits, known as the type I and type II receptors. Both subunits contain a serine/threonine kinase domain—a unique feature among transmembrane receptors in animal cells. Other TGF-binding proteins include the type III receptors and endoglin. The nonsignaling role of the type III receptors is shared by other abundant proteoglycan cytokine receptors. Endoglin is specifically expressed in endothelial cells and mutated in patients with hereditary hemorrhagic telangiectasia. The type II receptors bind to TGF, but the type I receptors cannot do so alone. Thus, the function of constitutively activated type II receptors is to activate type I receptors. The type I receptors propagate the TGFsignal by phosphorylating the Smads. To date, eight types of Smad proteins have been identified in mammals. These Smad proteins are classified into three functional and structural categories: receptor-regulated Smad (R

An Adult Mouse Thyroid Side Population Cell Line that Exhibits Enriched Epithelial–Mesenchymal Transition

BACKGROUND Studies of thyroid stem/progenitor cells have been hampered due to the small organ size and lack of tissue, which limits the yield of these cells. A continuous source that allows the study and characterization of thyroid stem/progenitor cells is desired to push the field forward. METHOD A cell line was established from Hoechst-resistant side population cells derived from mouse thyroid that were previously shown to contain stem/progenitor-like cells. Characterization of these cells were carried out by using in vitro two- and three-dimensional cultures and in vivo reconstitution of mice after orthotopic or intravenous injection, in conjunction with quantitative reverse transcription polymerase chain reaction, Western blotting, immunohisto(cyto)chemistry/immunofluorescence, and RNA seq analysis. RESULTS These cells were named SPTL (side population cell-derived thyroid cell line). Under low serum culturing conditions, SPTL cells expressed the thyroid differentiation marker NKX2-1, a transcription factor critical for thyroid differentiation and function, while no expression of other thyroid differentiation marker genes were observed. SPTL cells formed follicle-like structures in Matrigel® cultures, which did not express thyroid differentiation marker genes. In mouse models of orthotopic and intravenous injection, the latter following partial thyroidectomy, a few SPTL cells were found in part of the follicles, most of which expressed NKX2-1. SPTL cells highly express genes involved in epithelial-mesenchymal transition, as demonstrated by RNA seq analysis, and exhibit a gene-expression pattern similar to anaplastic thyroid carcinoma. CONCLUSION These results demonstrate that SPTL cells have the capacity to differentiate into thyroid to a limited degree. SPTL cells may provide an excellent tool to study stem cells, including cancer stem cells of the thyroid.

Abstract 20: GIST-specific markers identified by comprehensive gene expression profiles.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasm of the gastrointestinal tract and highly express c-kit. Although surgical removal and molecularly targeted therapy are known to be effective, specific markers other than c-kit would be beneficial for histopathological classification, risk assessment, and treatment decision. To this end, we conducted a DNA-microarry analysis using approximately 900 tumor tissues obtained in Fukushima Medical University Hospital from 2007 to present. The database contained more than 30 kinds of tumors including 10 cases of GIST. We searched ∼32,000 genes on the array that showed expression ratio of >2.0 in GIST samples compared to others, and finally extracted 54 GIST-specific genes. In this study, we identified that cathepsin L mRNA is highly expressed in GISTs. Moreover, immunohistochemical analysis of 43 GIST specimens revealed that 86.0% (n = 37) were cathepsin-L positive, and this positivity was significantly correlated with c-kit positivity. From these results, we conclude that cathepsin L is highly expressed in GISTs. Citation Format: Manabu Iwadate, Kohtaro Miyamoto, Emi Ito, Jun-ichi Imai, Naoki Sawada, Izumi Nakamura, Shinji Ohki, Shinya Watanabe, Satoshi Waguri, Seiichi Takenoshita. GIST-specific markers identified by comprehensive gene expression profiles. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 20. doi:10.1158/1538-7445.AM2013-20

Abstract 438: Establishment of MDA-MB231-derived breast cancer cell lines possessing a higher metastatic activity and a resistance to radiation.

Background and purpose: Metastatic cancer tissues might have characteristics different from their original primary regions, which could affect strategies for radiotherapy. To investigate properties of metastatic cancers and their radiosensitivities, we established MDA-MB231-derived breast cancer cell lines, which were prone to cause metastasis when transplanted into mouse. Methods: A breast cancer cell line, MDA-MB231 expressing pGL4.5/luciferase were injected into nude mice via left ventricle or mammary gland tissue to form metastatic lesions. The metastatic cells in lymph nodes, bone, and lung were detected by a bioluminescence imaging technique, which were excised, cultured, and transplanted into the next mice in the same way. Such transplantation procedures were repeated 3 times to establish organ-oriented metastasis-prone cell lines. The different characteristics of these cells were examined by morphological analysis, and proliferation, invasion, and migration assays, as well as radiation sensitivity test. Result: There was no significant difference among cell lines in morphology and proliferative ability. All three metastasis-prone cell lines showed increased activity in migration and invasion compared to the parental cell line. Moreover, they showed lower sensitivities to radiation than the parental cell line, while no difference was found among the metastasis-prone cell lines. Conclusion: We have established new cell lines that could be used for the study of metastatic breast cancers in relation to the radiotherapy. Citation Format: Takamitsu Hara, Manabu Iwadate, Shuichi Shiratori, Kenji Gonda, Tatsuo Shimura, Yoshihiro Nakagami, Masahiko Shibata, Satoshi Waguri, Seiichi Takenoshita. Establishment of MDA-MB231-derived breast cancer cell lines possessing a higher metastatic activity and a resistance to radiation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 438. doi:10.1158/1538-7445.AM2013-438