Noriko Hanamura

Expression of fibronectin and tenascin-C mRNA by myofibroblasts, vascular cells and epithelial cells in human colon adenomas and carcinomas

To understand the mechanisms of tissue remodeling during cancer progression, it is important to know the type of cells that actively express extracellular matrix (ECM) proteins. Twenty‐nine adenocarcinomas, 5 adenomas and non‐neoplastic mucosa samples were therefore investigated to determine their fibronectin (FN) and tenascin‐C (TN‐C) expression using in situ hybridization (ISH) and immunohistochemical staining. In the non‐neoplastic mucosa, no mRNA signals were found. Two of the adenomas demonstrated positive signals in peri‐cryptal cells and the vessels. In the cancers, TN‐C and FN mRNAs were found in 86% and 96% of the total cases, respectively. The signals were mainly detected in myofibroblasts, labeled with α‐smooth muscle actin, in the cancer stroma. TN‐C mRNA‐positive cells were often observed in localized areas, such as in cancer stroma associated with invading edges and/or in host tissues surrounding the invading cancer front, but rarely in the center of the tumors. FN mRNA‐positive cells were more widely spread throughout the cancer stroma, although they were also frequently observed at invading edges. Vascular cells in cancer tissues were also labeled. In 10 specimens, cancer cells themselves expressed FN and/or TN‐C mRNA. Comparison with histo‐pathological findings revealed positive relationships between the degree of mRNA expression of FN and TN‐C and the depth of invasion as well as the frequency of metastasis to lymph nodes. The expression of FN and TN‐C by myofibroblasts, vascular cells and cancer cells could be important for the remodeling process of neoplastic tissues during cancer development and progression. Int. J. Cancer 73:10–15, 1997.

Co-expression of tenascin and fibronectin in epithelial and stromal cells of benign lesions and ductal carcinomas in the human breast

Tenascin (TN)‐C and fibronectin (FN), which are glycoproteins of the extracellular matrix (ECM), are up‐regulated in cancer tissues, including breast cancer. For assessment of their involvement in cancer invasion, it is important to know which cells are responsible for their production and secretion. The distribution of cells expressing TN and FN mRNAs in benign and malignant human breast tissues was therefore analysed by in situ hybridization, using digoxigenin‐labelled cRNA probes, in addition to demonstrating the proteins immunohistochemically. Both mRNAs were expressed in epithelial cancer as well as in stromal cells in a large fraction of the tumours, with co‐expression in individual cells. In cancers with intraductal components and/or those consisting of large nests, the mRNAs were more often expressed in the cancer than in the stromal cells. In scirrhous carcinomas, in contrast, the stromal cells were almost always positive for TN and FN mRNAs, while the cancer cells only rarely exhibited TN or FN expression. In benign lesions including adenosis, fibroadenoma and intraductal papilloma, the expression patterns also varied. These findings indicate that TN and FN co‐expressed by cancer cells and stromal cells are probably involved in the intraductal extension and early invasion of cancer cells and in the remodelling of cancer stroma.

Tenascin C Induces Epithelial-Mesenchymal Transition–Like Change Accompanied by SRC Activation and Focal Adhesion Kinase Phosphorylation in Human Breast Cancer Cells

Tenascin C (TNC) is an extracellular matrix glycoprotein up-regulated in solid tumors. Higher TNC expression is shown in invading fronts of breast cancer, which correlates with poorer patient outcome. We examined whether TNC induces epithelial-mesenchymal transition (EMT) in breast cancer. Immunohistochemical analysis of invasive ductal carcinomas showed that TNC deposition was frequent in stroma with scattered cancer cells in peripheral margins of tumors. The addition of TNC to the medium of the MCF-7 breast cancer cells caused EMT-like change and delocalization of E-cadherin and β-catenin from cell-cell contact. Although amounts of E-cadherin and β-catenin were not changed after EMT in total lysates, they were increased in the Triton X-100-soluble fractions, indicating movement from the membrane into the cytosol. In wound healing assay, cells were scattered from wound edges and showed faster migration after TNC treatment. The EMT phenotype was correlated with SRC activation through phosphorylation at Y418 and phosphorylation of focal adhesion kinase (FAK) at Y861 and Y925 of SRC substrate sites. These phosphorylated proteins colocalized with αv integrin-positive adhesion plaques. A neutralizing antibody against αv or a SRC kinase inhibitor blocked EMT. TNC could induce EMT-like change showing loss of intercellular adhesion and enhanced migration in breast cancer cells, associated with FAK phosphorylation by SRC; this may be responsible for the observed promotion of TNC in breast cancer invasion.

Effect of Pancreatic Juice Reflux into Biliary Tract on N-Nitrosobis(2-oxopropyl)amine (BOP)-Induced Biliary Carcinogenesis in Syrian Hamsters

To elucidate the possible role of pancreaticjuice reflux into the biliary tract in promoting thedevelopment of biliary carcinoma, Syrian hamsters weresubjected to cholecystoduodenostomy and ligation of the distal end of the common duct and thensubcutaneously injected withN-nitrosobis(2-oxopropyl)amine (BOP) (experimentalgroup). The incidences of gallbladder carcinoma andextrahepatic bile duct carcinoma in the experimental group wassignificantly higher than in the sham-operated group (P< 0.01, P < 0.05). The proliferating cell nuclearantigen (PCNA) labeling indices of both regionsgradually increased with time, and were significantlyhigher in the experimental group at weeks 9 and 16 thanin the sham-operated group at the same time. Trypsin andphospholipase A2 (PLA2) activitiesin bile and tissue levels of superoxide dismutase (SOD) inthe gallbladder and extrahepatic bile ducts were higherin the experimental group than in the sham-operatedgroup. These findings suggest that the carcinogenic effect of BOP was enhanced in biliaryepithelium that had proliferated in response to and/orhad been injured by activated pancreatic enzymesrefluxing into the biliary tract and then increased freeradical activity, leading to a high frequency ofcarcinoma development in the biliary tract.

Binding of αvβ1 and αvβ6 integrins to tenascin-C induces epithelial–mesenchymal transition-like change of breast cancer cells

Tenascin-C (TNC), a large hexameric extracellular glycoprotein, is a pleiotropic molecule with multiple domains binding to a variety of receptors mediating a wide range of cellular functions. We earlier reported that TNC induces epithelial–mesenchymal transition (EMT)-like change in breast cancer cells. In the present study, we clarified TNC receptor involvement in this process. Among integrins previously reported as TNC receptors, substantial expression of αv, α2, β1 and β6 subunits was detected by quantitative PCR and immunoblotting in MCF-7 cells. Integrin β6 mRNA was remarkably upregulated by transforming growth factor (TGF)-β1 treatment, and protein expression was prominently increased by additional exposure to TNC. Immunofluorescent labeling demonstrated integrin αvβ6 accumulation in focal adhesions after TNC treatment, especially in combination with TGF-β1. The α2 and β1 subunits were mainly localized at cell–cell contacts, αv being found near cell cluster surfaces. Immunoprecipitation showed increase in αvβ1 heterodimers, but not α2β1, after TNC treatment. Activated β1 subunits detected by an antibody against the Ca2+-dependent epitope colocalized with αv in focal adhesion complexes, associated with FAK phosphorylation at tyrosine 925. Neutralizing antibodies against αv and β1 blocked EMT-like change caused by TNC alone. In addition, anti-αv and combined treatment with anti-β1 and anti-αvβ6 inhibited TGF-β1/TNC-induced EMT, whereas either of these alone did not. Integrin subunits αv, β1 and β6, but not α2, bound to TNC immobilized on agarose beads in a divalent cation-dependent manner. Treatments with neutralizing antibodies against β1 and αvβ6 reduced αv subunit bound to the beads. Immunohistochemistry of these receptors in human breast cancer tissues demonstrated frequent expression of β6 subunits in cancer cells forming scattered nests localized in TNC-rich stroma. These findings provide direct evidence that binding of αvβ6 and αvβ1 integrins to TNC as their essential ligand induces EMT-like change in breast cancer cells.

Breast-Volume Displacement Using an Extended Glandular Flap for Small Dense Breasts

We defined the glandular flap including fat in the subclavicular area as an extended glandular flap, which has been used for breast-conserving reconstruction in the upper portion of the breast. Indication. The excision volume was 20% to 40% of the breast volume, and the breast density was dense. Surgical Technique. The upper edge of the breast at the subclavicular area was drawn in the standing position before surgery. After partial mastectomy, an extended glandular flap was made by freeing the breast from both the skin and the pectoralis fascia up to the preoperative marking in the subclavicular area. It is important to keep the perforators of the internal mammary artery and/or the branches of the lateral thoracic artery intact while making the flap. Results. Seventeen patients underwent remodeling using an extended glandular flap. The cosmetic results at 1 year after the operation: excellent in 11, good in 1, fair in 3, and poor in 2. All cases of unacceptable outcome except one were cases with complications, and more than 30% resection of moderate or large size breasts did not obtain an excellent result for long-term followup. Conclusion. This technique is useful for performing the breast-conserving reconstruction of small dense breasts.

Oncoplastic Technique Combining an Adipofascial Flap with an Extended Glandular Flap for the Breast-Conserving Reconstruction of Small Dense Breasts

We introduce a method combining two oncoplastic techniques for breast-conserving reconstruction. The procedure is as follows: first, an extended glandular flap is made by undermining the breast from both the skin and the pectoralis fascia to the upper edge of the breast at the subclavicular area. After modeling the breast mound with the extended glandular flap, an inframammary adipofascial flap is made. The flap is reflected back to the breast area remodeled using the extended glandular flap. After reshaping the breast, the inframammary line is then re-shaped. This method is indicated for patients with breast cancer in the outer portion of the breast, who have small dense breasts, and have undergone a large excision of about 40% of their breast volume. We treated four patients, all of whom had either excellent or good cosmetic results with no fat necrosis.

Abdominal advancement flap as oncoplastic breast conservation: report of seven cases and their cosmetic results.

An abdominal advancement flap (AAF) is a flap that pulls the elevated abdominal skin up and creates the shape of the inferior portion of the breast by making a neo-inframammary fold. Seven patients underwent remodeling using an AAF or a method combining an AAF with other volume displacement techniques after partial mastectomy. The excision volume ranged from 15% to 35%. AAF with only mobilization of the gland flaps was performed in two cases, with lateral mammoplasty in one case, with the round block technique (RBT) in one case, with a modified RBT in one case, and with medial mammoplasty in two cases. Although one patient treated with a RBT had a partial blood-flow insufficiency of the nipple-areola complex, it improved with conservative treatment. The cosmetic results were found to be excellent in three cases, good in three, and fair in one case.