Shinji Mii

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.

Sprouty2 regulates growth and differentiation of human neuroblastoma cells through RET tyrosine kinase

The Sprouty (SPRY) family of proteins includes important regulators of downstream signaling initiated by receptor tyrosine kinases. In the present study, we investigated the role of SPRY proteins in intracellular signaling via the RET receptor tyrosine kinase activated by glial cell line‐derived neurotrophic factor (GDNF). Expression of SPRY1, SPRY2, SPRY3 and SPRY4 in HEK293T cells transfected with RET and GDNF receptor family α1 (GFRα1) genes significantly reduced sustained ERK activation as well as ELK‐1 activation. Because expression of SPRY2 was efficiently induced by GDNF in TGW human neuroblastoma cells expressing RET and GFRα1, we further investigated the role of SPRY2 in the growth and differentiation of TGW cells. Expression of wild‐type SPRY2 (WT‐SPRY2) decreased the growth of TGW cells. In contrast, expression of a dominant negative form of SPRY2 (MT‐SPRY2, with a mutated tyrosine residue) enhanced cell proliferation. In addition, expression of WT‐SPRY2 reduced GDNF‐dependent neurite outgrowth of TGW cells, whereas expression of MT‐SPRY2 enhanced it. Taken together, our results suggest that SPRY2 regulates GDNF‐dependent proliferation and differentiation of TGW neuroblastoma cells mediated by RET tyrosine kinase. (Cancer Sci 2007; 98: 815–821)

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.

Akt–Girdin Signaling in Cancer-Associated Fibroblasts Contributes to Tumor Progression

PI3K-Akt signaling is critical for the development, progression, and metastasis of malignant tumors, but its role in the tumor microenvironment has been relatively little studied. Here, we report that the Akt substrate Girdin, an actin-binding protein that regulates cell migration, is expressed and activated by Akt phosphorylation in cancer-associated fibroblasts (CAF) and blood vessels within the tumor microenvironment. Lewis lung tumors grafted into mice defective in Akt-mediated Girdin phosphorylation (SA transgenic mice) exhibited a decrease in both CAF infiltration and tumor growth, compared with wild-type (WT) host control animals. Contrasting with the findings of other studies, we found that Akt-dependent phosphorylation of Girdin was not a rate-limiting step in the growth of endothelial cells. In addition, Lewis lung tumors displayed limited outgrowth when cotransplanted with CAF derived from tumor-bearing SA transgenic mice, compared with CAF derived from tumor-bearing WT mice. Collectively, our results revealed a role for Akt-mediated Girdin phosphorylation in CAF during tumor progression, highlighting the need to inhibit Akt function in both tumor cells and cells that comprise the tumor microenvironment.

The REV7 Subunit of DNA Polymerase ζ Is Essential for Primordial Germ Cell Maintenance in the Mouse

Background: Biological significance of REV7 in mouse development has not been elucidated. Results: REV7-deficient mice show germ cell aplasia at birth in both sexes, and primordial germ cells (PGCs) were lost because of apoptosis during migration at an early embryonic stage. Conclusion: REV7 is essential for PGC maintenance in the mouse. Significance: REV7 is a novel regulator of PGC survival. REV7 (also known as MAD2L2 and MAD2B) is involved in DNA repair, cell cycle regulation, gene expression, and carcinogenesis. In vitro studies show that REV7 interacts with several proteins and regulates their function. It has been reported that human REV7 is highly expressed in the adult testis by Northern blot analysis. However, the significance of REV7 in mammalian development has not been elucidated. Here, we present analyses of REV7-deficient (Rev7−/−) mice to clarify the significance of Rev7 in mouse development. In WT mice (Rev7+/+), Rev7 expression was ubiquitously observed in the embryo and confined to germ cells in the testes after birth. Rev7−/− mice exhibited growth retardation and a partial embryonic lethal phenotype. Mice that survived to adulthood were infertile in both sexes and showed germ cell aplasia in the testes and ovaries. Analyses of Rev7−/− embryos revealed that primordial germ cells (PGCs) were present at embryonic day 8.5 (E8.5). However, progressive loss of PGCs was observed during migration, and PGCs were absent in the genital ridges at E13.5. An increase of apoptotic cells was detected not only among PGCs but also in the forebrain of the Rev7−/− embryo, whereas cell proliferation was unaffected. Moreover, DNA damage accumulation and increased levels of histone methylation were detected in Rev7−/− embryos, and expression of Oct4 and Nanog was deregulated by REV7 deficiency at E8.5. These findings indicate that Rev7 is essential for PGC maintenance by prevention of apoptotic cell death in the mouse.

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.

Protective Role of Gipie, a Girdin Family Protein, in Endoplasmic Reticulum Stress Responses in Endothelial Cells

The Gipie/GRP78 interaction serves as a molecular switch for the regulation of the apoptotic signaling pathway, which appears to contribute to the protection of endothelial cells from endoplasmic reticulum stress-induced apoptosis.

TRIM27/MRTF-B-Dependent Integrin β1 Expression Defines Leading Cells in Cancer Cell Collectives

For collective invasion, cancer cells form cohesive groups comprised of leading cells (LCs) at the forefront and following cells (FCs) at the rear. However, the molecular mechanisms that define LCs and FCs remain elusive. Here, we demonstrated that LCs, but not FCs, upregulated the expression of integrin β1 after the loss of intercellular adhesion. The LC-specific expression of integrin β1 was posttranscriptionally regulated by the TRIM27/MRTF-B complex in response to the loss of intercellular adhesion, thereby regulating the stability and translation of integrin β1 mRNA via microRNA-124 in LCs. Accordingly, depletion of TRIM27 and MRTF-B abrogated the upregulation of integrin β1 in LCs and blocked the invasion of cancer cell groups in vitro and in vivo. Therefore, our findings revealed that the specific function of LCs was defined by intrinsic mechanisms related to the presence of the cells free surface, providing insights into the regulation of intratumor heterogeneity.

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.