Toshiaki Yoshioka

β4 Integrin Amplifies ErbB2 Signaling to Promote Mammary Tumorigenesis

Amplification of the ErbB2 locus, which encodes a receptor tyrosine kinase, is common in aggressive breast tumors and correlates with poor prognosis. The mechanisms underlying ErbB2-mediated breast carcinoma progression remain incompletely defined. To examine the role of the signaling and cell-adhesion receptor beta 4 integrin during ErbB2-mediated tumorigenesis, we introduced a targeted deletion of the beta 4 signaling domain into a mouse model of ErbB2-induced mammary carcinoma. Loss of beta 4 signaling suppresses mammary tumor onset and invasive growth. Ex vivo studies indicate that beta 4 forms a complex with ErbB2 and enhances activation of the transcription factors STAT3 and c-Jun. STAT3 contributes to disruption of epithelial adhesion and polarity, while c-Jun is required for hyperproliferation. Finally, deletion of the beta 4 signaling domain enhances the efficacy of ErbB2-targeted therapy. These results indicate that beta 4 integrin promotes tumor progression by amplifying ErbB2 signaling and identify beta 4 as a potential target for molecular therapy of breast cancer.

Targeted Deletion of the Integrin β4 Signaling Domain Suppresses Laminin-5-Dependent Nuclear Entry of Mitogen-Activated Protein Kinases and NF-κB, Causing Defects in Epidermal Growth and Migration

ABSTRACT The α6β4 integrin—a laminin-5 receptor—mediates assembly of hemidesmosomes and recruitment of Shc and phosphoinositide 3-kinase through the unique cytoplasmic extension of β4. Mice carrying a targeted deletion of the signaling domain of β4 develop normally and do not display signs of skin fragility. The epidermis of these mice contains well-structured hemidesmosomes and adheres stably to the basement membrane. However, it is hypoplastic due to reduced proliferation of basal keratinocytes and undergoes wound repair at a reduced rate. Keratinocytes from β4 mutant mice undergo extensive spreading but fail to proliferate and migrate in response to epidermal growth factor (EGF) on laminin-5. EGF causes significant phosphorylation of extracellular signal-regulated kinase (ERK) and Jun N-terminal protein kinase (JNK) and phosphorylation and degradation of IκB in β4 mutant cells adhering to laminin-5. Unexpectedly, however, ERK, JNK, and NF-κB remain in the cytoplasm in β4 mutant cells on laminin-5, whereas they enter effectively into the nucleus in the same cells on fibronectin or in wild-type cells on both matrix proteins. Inhibitor studies indicate that α6β4 promotes keratinocyte proliferation and migration through its effect on NF-κB and P-JNK. These findings provide evidence that β4 signaling promotes epidermal growth and wound healing through a previously unrecognized effect on nuclear translocation of NF-κB and mitogen-activated protein kinases.

Transdifferentiation of Mature Rat Hepatocytes into Bile Duct-Like Cells in Vitro

We investigated the mechanism of phenotypic plasticity of hepatocytes in a three-dimensional organoid culture system, in which hepatocytic spheroids were embedded within a collagen gel matrix. Hepatocytes expressed several bile duct markers including cytokeratin (CK) 19 soon after culture and underwent branching morphogenesis within the matrix in the presence of insulin and epidermal growth factor. Cultured hepatocytes did not express Delta-like, a specific marker for oval cells and hepatoblasts. Furthermore, hepatocytes isolated from c-kit mutant rats (Ws/Ws), which are defective in proliferation of oval cells, showed essentially the same phenotypic changes as those isolated from control rats. The bile duct-like differentiation of hepatocytes was associated with increased expression of Jagged1, Jagged2, Notch1, and several Notch target genes. CK19 expression and branching morphogenesis were inhibited by dexamethasone, a mitogen-activated protein kinase kinase 1 (MEK1) inhibitor (PD98059), and a phosphatidyl inositol 3-kinase inhibitor (LY294002). After being cultured for more than 3 weeks within the gels, hepatocytes transformed into ductular structures surrounded by basement membranes. Our results suggest that hepatocytes might have the potential to transdifferentiate into bile duct-like cells without acquiring a stem-like phenotype and that this is mediated through specific protein tyrosine phosphorylation pathways.

A highly specific isolation of rat sinusoidal endothelial cells by the immunomagnetic bead method using SE-1 monoclonal antibody

BACKGROUND/AIMS To develop a specific isolation method of hepatic sinusoidal endothelial cells (SEC), we applied the immunomagnetic method using a monoclonal antibody (SE-1) that recognizes a membranous antigen expressed only in rat SEC. METHODS Cells were isolated by incubating mixed non-parenchymal cells, which were obtained by collagenase digestion of the liver, with SE-1-conjugated superparamagnetic polystyrene beads. The conventional Percoll method was also performed in parallel to compare with the immunomagnetic method. The isolated cells were cultured on glass coverslips coated with type I collagen in the presence of various growth factors for 6 days. RESULTS Approximately 98% of the isolated cells were positive for SE-1 and the contamination of Kupffer cells or stellate cells was less than 1%. The purity was significantly better than that obtained by the Percoll method. The cultured cells showed typical SEC features, such as sieve plates and uptake of acetylated low-density lipoprotein. Although the cells continuously underwent apoptotic cell death after 2 days, they started robust cell growth after 3 days and were well maintained during the culture period. CONCLUSIONS Our simple and specific isolation method enables us to culture SEC with high purity and should be useful for the biological analysis of SEC.

Excision Repair Cross-Complementing Group 1 May Predict the Efficacy of Chemoradiation Therapy for Muscle-Invasive Bladder Cancer

Purpose: Chemoradiation therapy (CRT) is now widely recognized as bladder-preserving therapy for muscle-invasive bladder cancer (MIBC). However, some patients who fail CRT may miss the chance to be cured by cystectomy. Therefore, it is important to select patients with MIBC who are expected to have a good response to CRT. Several reports indicate that the excision repair cross-complementing group 1 (ERCC1) gene is associated with resistance to cisplatin and radiation therapy. In this study, we examined the correlation between ERCC1 and CRT in vitro and in vivo in bladder cancer. Experimental Design: Bladder cancer cell lines T24, 5637, Cl8-2 (multidrug-resistant subline of T24), and CDDP10-3 (cisplatin-resistant subline of T24) were used for in vitro assays to measure ERCC1 expression level and growth inhibition with cisplatin or ionizing radiation (IR). We then examined by immunohistochemistry that whether ERCC1 nuclear staining correlates with the efficacy of CRT using cisplatin in 22 patients with MIBC. Results: Cl8-2 cells expressed ERCC1 mRNA 5.96-fold higher than did T24. Cl8-2 and CDDP10-3 were more resistant to cisplatin or IR than was T24. Resistance to IR, but not to cisplatin, was removed by suppressing ERCC1 using siRNA in both Cl8-2 and CDDP10-3 cells. In immunohistochemistry with ERCC1, 6 of 8 positive cases did not have complete response to CRT, whereas 12 of 14 negative cases had complete response. Sensitivity and specificity were 75% and 85.7%, respectively (P = 0.008). Conclusion: Although further study is needed, ERCC1 expression level may predict the efficacy of CRT for MIBC. Clin Cancer Res; 17(8); 2561–9. ©2010 AACR.

Immunohistochemical characterization of microglia in Nasu-Hakola disease brains

Nasu‐Hakola disease (NHD) is a rare autosomal recessive disorder, characterized by progressive presenile dementia and formation of multifocal bone cysts, caused by genetic mutations of DNAX‐activation protein 12 (DAP12) or triggering receptor expressed on myeloid cells 2 (TREM2). TREM2 and DAP12 constitute a receptor/adapter signaling complex expressed on osteoclasts, dendritic cells (DC), macrophages and microglia. Previous studies using knockout mice and mouse brain cell cultures suggest that a loss‐of‐function of DAP12/TREM2 in microglia plays a central role in the neuropathological manifestation of NHD. However, there exist no immunohistochemical studies that focus attention on microglia in NHD brains. To elucidate a role of microglia in the pathogenesis of NHD, we searched NHD‐specific biomarkers and characterized their expression on microglia in NHD brains. Here, we identified allograft inflammatory factor 1 (AIF1, Iba1) and sialic acid binding Ig‐like lectin 1 (SIGLEC1) as putative NHD‐specific biomarkers by bioinformatics analysis of microarray data of NHD DC. We studied three NHD and eight control brains by immunohistochemistry with a panel of 16 antibodies, including those against Iba1 and SIGLEC1. We verified the absence of DAP12 expression in NHD brains and the expression of DAP12 immunoreactivity on ramified microglia in control brains. Unexpectedly, TREM2 was not expressed on microglia but expressed on a small subset of intravascular monocytes/macrophages in control and NHD brains. In the cortex of NHD brains, we identified accumulation of numerous Iba1‐positive microglia to an extent similar to control brains, while SIGLEC1 was undetectable on microglia in all the brains examined. These observations indicate that human microglia in brain tissues do not express TREM2 and DAP12‐deficient microglia are preserved in NHD brains, suggesting that the loss of DAP2/TREM2 function in microglia might not be primarily responsible for the neuropathological phenotype of NHD.

β4 Integrin signaling induces expansion of prostate tumor progenitors

The contextual signals that regulate the expansion of prostate tumor progenitor cells are poorly defined. We found that a significant fraction of advanced human prostate cancers and castration-resistant metastases express high levels of the β4 integrin, which binds to laminin-5. Targeted deletion of the signaling domain of β4 inhibited prostate tumor growth and progression in response to loss of p53 and Rb function in a mouse model of prostate cancer (PB-TAg mice). Additionally, it suppressed Pten loss-driven prostate tumorigenesis in tissue recombination experiments. We traced this defect back to an inability of signaling-defective β4 to sustain self-renewal of putative cancer stem cells in vitro and proliferation of transit-amplifying cells in vivo. Mechanistic studies indicated that mutant β4 fails to promote transactivation of ErbB2 and c-Met in prostate tumor progenitor cells and human cancer cell lines. Pharmacological inhibition of ErbB2 and c-Met reduced the ability of prostate tumor progenitor cells to undergo self-renewal in vitro. Finally, we found that β4 is often coexpressed with c-Met and ErbB2 in human prostate cancers and that combined pharmacological inhibition of these receptor tyrosine kinases exerts antitumor activity in a mouse xenograft model. These findings indicate that the β4 integrin promotes prostate tumorigenesis by amplifying ErbB2 and c-Met signaling in tumor progenitor cells.

Cytoplasmic accumulation of connexin32 protein enhances motility and metastatic ability of human hepatoma cells in vitro and in vivo.

Connexins have long been believed to suppress tumour development during carcinogenesis by exerting gap junctional intercellular communication (GJIC). Although GJIC is abrogated in hepatocellular carcinoma (HCC), connexin32 (Cx32) protein tends to remain expressed in cytoplasm, but not in cell–cell contact areas; thus, it is incapable of forming gap junctions. Hypothesising that cytoplasmic Cx32 protein that has accumulated in HCC should have its proper functions, which are independent of GJIC, we established an inducible expression system of Cx32 in human HuH7 HCC cells, which were unable to support the formation of Cx32‐mediated gap junctions, so that Cx32 protein could be overexpressed by doxycycline (Dox) withdrawal. Although the established clone HuH7 Tet‐off Cx32 cells exhibited a 4‐fold increase in Cx32 expression after Dox withdrawal, none of them were dye‐coupled, and Cx32 protein was retained in the Golgi apparatus. However, the proliferation rate of the HuH7 Tet‐off Cx32 cells was significantly higher in the Dox‐free medium than in the Dox‐supplemented one. Transwell assays also revealed that Dox withdrawal enhanced serum‐stimulated motility and invasiveness into Matrigel of the HuH7 Tet‐off Cx32 cells. Furthermore, when HuH7 Tet‐off Cx32 cells were xenografted into the liver of SCID mice, only the mice to which no Dox was administered developed metastatic lesions, indicating that overexpression of cytoplasmic Cx32 protein induced metastasis of HuH7 cells. Our results suggest that, while Cx32‐mediated GJIC suppresses the development of HCCs, cytoplasmic Cx32 protein exerts effects favourable for HCC progression, such as invasion and metastasis, once the cells have acquired a malignant phenotype.

Pathological Significance of Intracytoplasmic Connexin Proteins: Implication in Tumor Progression

A considerable amount of evidence has established that gap junctional intercellular communication (GJIC) suppresses tumor development by halting the stage of tumor promotion. Consistently, GJIC is downregulated in tumors. The downregulation of GJIC is caused by not only the reduced expression level of connexin proteins but also their aberrant cytoplasmic localization. Although it has long been thought that cytoplasmic localization of connexin proteins is merely one of the mechanisms of the downregulation of GJIC, careful studies with human tumor samples have indicated that the expression level of intracytoplasmic connexin proteins correlates well with the grade of malignancy and the progression stage of tumors. Hypothesizing that intracytoplasmic connexin proteins should have their proper functions and that their increase should facilitate tumor progression such as cell migration, invasion and metastasis, we examined the effects of overexpressed connexin32 (Cx32) protein on the phenotype of human HuH7 hepatoma cells, which express a basal level of endogenous Cx32 only in cytoplasm. The cells were retrovirally transduced with the Tet-off Cx32 construct so that withdrawal of doxycycline from the culture medium could induce overexpression of Cx32 protein in cytoplasm. Even when overexpressed, Cx32 protein was retained in cytoplasm, i.e., Golgi apparatuses, and did not induce GJIC. However, overexpression of Cx32 protein in cytoplasm enhanced both the motility and the invasiveness of HuH7 cells and induced metastasis when the cells were xenografted into SCID mice. Taken together, cytoplasmic accumulation of connexin proteins may exert effects favorable for tumor progression.

Homotypic Adhesion through Carcinoembryonic Antigen Plays a Role in Hepatic Metastasis Development

We established a cell line with high metastatic potential to the liver (LS‐LM4) after four successive repetitions of splenic injection of liver‐metastatic cells in SCID mice. This cell line strongly expressed CEA and showed increased homotypic adhesion as compared with the parent cell line (LS174T). To examine the role of CEA in the increased homotypic adhesion, LS‐LM4 cells were treated with anti‐CEA antibody and subjected to an in vitro adhesion and aggregation assay. Further, to study the role of CEA in the hepatic metastasis of cells with high metastatic potential, LS‐LM4 cells were treated with anti‐CEA antibody, and the inhibition of hepatic metastasis after splenic injection in vivo was examined. There was a 62% decrease in the homotypic adhesion of anti‐CEA antibody‐treated (100 μg/ml) LS‐LM4 cells under a Ca2+‐free condition as compared with the control (P<0.01). Anti‐CEA antibody (100 μg/ml) inhibited cell aggregation under a Ca2+‐free condition (P<0.05). Treatment with anti‐E‐cadherin antibody (60 μ/ml) plus anti‐CEA antibody (100 μg/ml) inhibited cell aggregation more potently than anti‐E‐cadherin antibody treatment alone in the presence of Ca2+. In vivo, there was a 75% decrease in the number of hepatic metastatic nodules in the G125 anti‐CEA antibody‐treated group as compared with the control group (P<0.01). Similarly, there was a 40% decrease in the diameter of metastatic nodules and there was a 90% decrease in total tumor volume of hepatic metastasis in the G125 anti‐CEA antibody‐treated group as compared with the control (P<0.01). These results suggest that increased metastatic potential to the liver is at least partly due to increased homotypic binding mediated by CEA.