Sumitaka Hagiwara

CD109 expression in basal‐like breast carcinoma

Breast cancer can be classified into several subtypes based on gene expression profiling. Basal‐like breast carcinoma (BLC) has a triple negative phenotype, that is, the subtype lacks the estrogen receptor (ER), progesterone receptor (PgR) and human epidermal growth factor receptor 2 (HER2). It has been recently reported that CD109, a glycosylphosphatidylinositol (GPI)‐anchored cell surface protein, is a new breast myoepithelial marker. In the present study CD109 expression was investigated in invasive ductal carcinomas (IDC) of the breast on immunohistochemistry. Eighty‐eight formalin‐fixed, paraffin‐embedded breast carcinoma sections were immunostained with anti‐CD109, anti‐cytokeratin 5/6 (CK5/6), anti‐calponin, anti‐vimentin and anti‐p63 antibodies. CD109 expression was detected in 18 of 30 basal‐like breast carcinomas (BLC) but not in other types of 53 IDC (non‐BLC) that were positive for ER, PgR and/or HER2. The percentage of CD109‐positive tissues (60%) in BLC was similar to that of CK5/6 (63%) and higher than that of other myoepithelial markers including p63 (23%), calponin (33%) and vimentin (33%). Statistical analysis indicated that the CD109‐positive group in BLC, but not the CK5/6‐positive group in BLC, was associated with reduced fat invasion (P < 0.05). These findings indicate that CD109 is a useful diagnostic marker for BLC and that CD109 expression may affect biological properties of cancer cells.

Up-regulation of CD109 expression is associated with carcinogenesis of the squamous epithelium of the oral cavity

CD109 is a glycosylphosphatidylinositol (GPI)–anchored glycoprotein whose expression is up‐regulated in squamous cell carcinomas (SCCs) of the lung, esophagus, and uterus. The purpose of this study was to evaluate CD109 expression in oral tumors, including premalignant lesions, and to assess the clinical application of CD109 in oral cancer. CD109 expression in oral normal and tumor tissues from 124 patients was examined by immunohistochemical staining with anti‐CD109 antibody, and significant relations between clinical features and CD109 expression were statistically assessed. We found that high levels of CD109 expression were frequently detected in SCCs and premalignant lesions of the oral cavity, but not in normal squamous epithelia. The CD109 expression level was higher in well‐differentiated SCCs than in poorly differentiated SCCs. Furthermore, premalignant lesions highly expressing CD109 showed higher risk to progress to SCCs. Oral SCC cell lines overexpressing CD109 exhibited accelerated cell growth in vitro compared with control cell lines. In addition, overexpression of CD109 impaired the transforming growth factor (TGF)–β1‐mediated suppression of cell growth. These findings suggest that CD109 plays a role in the development of oral cancers, and is a useful prognostic marker to predict malignant transformation of premalignant lesions. (Cancer Sci 2008; 99: 1916–1923)

CD109, a new marker for myoepithelial cells of mammary, salivary, and lacrimal glands and prostate basal cells.

The CD109 gene encodes a glycosylphosphatidylinositol (GPI)‐anchored cell surface protein. Herein it is shown that CD109 is highly expressed in myoepithelial cells of mammary, salivary, and lacrimal glands; and in prostate basal cells. The anti‐CD109 antibody generated by the authors was available for formalin‐fixed paraffin section, and it strongly stained myoepithelial cells and basal cells but not ductal, acinar, and secretory cells in these glands. CD109 expression was negative in examined breast ductal carcinomas and prostate adenocarcinomas. These findings indicate that CD109 is a useful marker for the diagnosis of invasive breast and prostate carcinomas.

Processing of CD109 by furin and its role in the regulation of TGF-|[beta]| signaling

CD109 is a glycosylphosphatidylinositol (GPI)-anchored glycoprotein, whose expression is upregulated in squamous cell carcinomas of the lung, esophagus, uterus and oral cavity. CD109 negatively regulates transforming growth factor (TGF)-β signaling in keratinocytes by directly modulating receptor activity. In this study, we further characterized CD109 regulation of TGF-β signaling and cell proliferation. We found that CD109 is produced as a 205 kDa glycoprotein, which is then processed in the Golgi apparatus into 180 kDa and 25 kDa proteins by furin (furinase). 180 kDa CD109 associated with GPI-anchored 25 kDa CD109 on the cell surface and was also secreted into the culture medium. To investigate whether furinase cleavage of CD109 is necessary for its biological activity, we mutated arginine 1273 in the CD109 furinase cleavage motif (amino acid 1270-RRRR-1273) to serine (R1273S). Interestingly, CD109 R1273S neither significantly impaired TGF-β signaling nor affected TGF-β-mediated suppression of cell growth, although it was expressed on the cell surface as a 205 kDa protein. Consistent with this finding, the 180 kDa and 25 kDa CD109 complex, but not CD109 R1273S, associated with the type I TGF-β receptor. These findings indicate that processing of CD109 into 180 kDa and 25 kDa proteins by furin, followed by complex formation with the type I TGF-β receptor is required for the regulation of TGF-β signaling in cancer cells and keratinocytes.

High-level expression of CD109 is frequently detected in lung squamous cell carcinomas.

CD109 is a glycosylphosphatidylinositol (GPI)‐anchored cell surface protein, which is a member of the α2‐macroglobulin/C3, C4, C5 family of thioester‐containing proteins. It has been reported that CD109 is expressed in a subset of hematopoietic cells, endothelial cells and several kinds of human tumors. Herein it is reported that the CD109 protein is preferentially expressed in lung squamous cell carcinomas compared with other types of lung carcinoma including adenocarcinomas, large cell carcinomas and small cell carcinomas. Immunohistochemical staining of surgically resected lung specimens using an anti‐CD109 antibody detected CD109 expression in basal cells of bronchial and bronchiolar epithelia and myoepithelial cells of bronchial secretary glands, but not in bronchial and bronchiolar apical epithelial cells and alveolar epithelial cells. Furthermore, the CD109 immunoreactivity was observed in squamous cell carcinomas at a high frequency compared with other types of lung carcinoma. Although the detailed function of CD109 protein is unclear, these results suggest that CD109 expression may play a role in the development of lung squamous cell carcinoma.

Correlation of pathological grade and tumor stage of urothelial carcinomas with CD109 expression.

Bladder cancer is one of the most common malignant diseases. Since a high‐rate of recurrence is a serious problem for early stage urothelial carcinomas, new strategies for the management of recurrent urothelial carcinomas have been explored. CD109 is a glycosylphosphatidylinositol‐anchored glycoprotein and is expressed in various cancer tissues, mainly squamous cell carcinomas. CD109 negatively controls transforming growth factor (TGF)‐β/Smad signaling in vitro. In this study, we analyzed the clinical significance of CD109 expression in bladder cancer using immunohistochemistry. Of 156 urothelial carcinoma tissues, 69.9% were positive for CD109, whereas CD109 was not expressed in seven normal bladder epithelia. CD109 expression was significantly higher in non‐muscle‐invasive (pTa+pT1) or low‐grade (G1+G2) tumors than in muscle‐invasive (pT2‐4) or high‐grade (G3) tumors, and was associated with cancer‐specific survival. Simultaneous immunostaining of CD109 and phosphorylated Smad2 showed an inverse immunoreactivity relationship between the two, suggesting that CD109 inhibits TGF‐β/Smad signaling in tumor tissues. Interestingly, CD109 was found to be highly expressed in the basal layer of non‐invasive urothelial carcinomas, and the expression pattern was similar to that of CD44, a marker of cancer stem cells. These findings suggest that CD109 is involved in bladder tumorigenesis and is a potential target for cancer immunotherapy.

Epidermal hyperplasia and appendage abnormalities in mice lacking CD109

CD109, a glycosylphosphatidylinositol-anchored glycoprotein, is highly expressed in several types of human cancer tissues, in particular, squamous cell carcinomas. In normal human tissues, human CD109 expression is limited to certain cell types including myoepithelial cells of the mammary, lacrimal, salivary, and bronchial glands and basal cells of the prostate and bronchial epithelium. Although CD109 has been reported to negatively regulate transforming growth factor-β signaling in keratinocytes in vitro, its physiologic role in vivo remains largely unknown. To investigate the function of CD109 in vivo, we generated CD109-deficient (CD109(-/-)) mice. Although CD109(-/-) mice were born normally, transient impairment of hair growth was observed. At histologic analysis, kinked hair shafts, ectatic hair follicles with an accumulation of sebum, and persistent hyperplasia of the epidermis and sebaceous glands were observed in CD109(-/-) mice. Immunohistochemical analysis revealed thickening of the basal and suprabasal layers in the epidermis of CD109(-/-) mice, which is where endogenous CD109 is expressed in wild-type mice. Although CD109 was reported to negatively regulate transforming growth factor-β signaling, no significant difference in levels of Smad2 phosphorylation was observed in the epidermis between wild-type and CD109(-/-) mice. Instead, Stat3 phosphorylation levels were significantly elevated in the epidermis of CD109(-/-) mice compared with wild-type mice. These results suggest that CD109 regulates differentiation of keratinocytes via a signaling pathway involving Stat3.

CD109 attenuates TGF-β1 signaling and enhances EGF signaling in SK-MG-1 human glioblastoma cells

CD109 is a glycosylphosphatidylinositol-anchored cell surface protein that is frequently detected in squamous cell carcinomas. CD109 is a negative regulator of TGF-β1 signaling in human keratinocytes, and the N-terminal fragment of CD109 secreted from cells after cleavage by the furin protease is important for modulating TGF-β1 signaling. Previously, we found that CD109 is expressed in human glioblastoma cells; however, the role of CD109 in glioblastoma cells is not established. Here, we describe the effects of CD109 in human glioblastoma cell lines. Three glioblastoma cell lines, SK-MG-1, U251MG and MG178, were tested and CD109 overexpression attenuated TGF-β1 signaling and enhanced EGF signaling in SK-MG-1, but not in U251MG or MG178. The N-terminal CD109 fragment in SK-MG-1 was hyperglycosylated compared with that in MG178 or U251MG. The conditioned medium of CD109-overexpressing SK-MG-1, containing the secreted N-terminal CD109, had a negative effect on TGF-β1 signaling in wild-type SK-MG-1 and MG178, whereas it did not show any effect on EGF signaling. In addition, cell surface CD109 interacts with EGF receptor in SK-MG-1 overexpressing CD109, and exhibited enhanced cell migration and invasion. These findings suggest that CD109 attenuates TGF-β1 signaling and enhances EGF signaling in SK-MG-1 cells and that the membrane-anchored CD109 may play major roles in the EGF signaling pathway.

Chemoradiotherapy for maxillary sinus adenoid cystic carcinoma using superselective intra-arterial infusion via a superficial temporal artery

The efficacy of concurrent intra‐arterial infusion chemoradiotherapy for adenoid cystic carcinoma (ACC) has been described in only a few reports. Herein, we report on 2 patients with unresectable ACC of the maxillary sinus treated with this approach.

Optimization of hyperthermia and dendritic cell immunotherapy for squamous cell carcinoma.

The aims of this study were to explore the feasibility of a novel combination therapy comprising hyperthermia (HT) and dendritic cell (DC) application for squamous cell carcinoma (SCC), and to optimize the conditions of this therapy. In vitro, the correlation between maturation of DCs by co-culture with SCCVII cells and HT was investigated. DCs did not mature in simple HT (43 °C for 30 min) with SCCVII cells. On the other hand, DC maturation occurred in additional mild HT (mHT: 41 °C for 30 min) with simple HT. To assess whether additional mHT was effective, in vivo combined treatment was performed using tumor-bearing C3H/HeJ mice. A more suppressive effect of tumor growth was observed, and cytotoxic T cell infiltration was significantly increased by adding mHT compared to conventional only simple HT with DCs. These phenomena also occurred in non-treated contralateral tumors as well as treated ones. Our data suggest that the combination of 43 °C preheated simple HT SCCVII tumors and additional 41 °C heat mHT promotes DC maturation, resulting in suppression of tumor growth systemically and lifetime prolongation in mice. A three-step process of additional mHT after local HT and intratumoral immature DC (iDC) injection could be a more effective and novel method for the treatment of SCC.