Tetsu Hayashida

Overexpression of leucine-rich repeat-containing G protein-coupled receptor 5 in colorectal cancer

Leucine‐rich repeat‐containing G protein‐coupled receptor 5 (LGR5) is a 7‐transmembrane receptor reportedly expressed in stem cells of the intestinal crypts and hair follicles of mice. Overexpression of LGR5 is observed in some types of cancer; however, there has been no specific assessment in colorectal tumorigenesis. We performed quantitative RT‐PCR for LGR5 expression in 37 representative cancer cell lines, and showed that LGR5 mRNA was frequently overexpressed in colon cancer cell lines. Moreover, LGR5 expression was higher in colon cancer cell lines derived from metastatic tumors compared with those from primary tumors. In clinical specimens, there was significant overexpression of LGR5 in 35 of 50 colorectal cancers (CRCs), and in seven of seven sporadic colonic adenomas, compared with matched normal mucosa. This suggests up‐regulation of LGR5 from the early stage of colorectal tumorigenesis. LGR5 expression showed marked variation among CRC cases and correlated significantly with lymphatic invasion, vascular invasion, tumor depth, lymph node metastasis, and tumor stage (IIIC vs. IIIB). In addition to cancer cells, crypt base columnar cells of the small intestine and colon were shown by in situ hybridization to express LGR5. This is the first report suggesting the involvement of LGR5, not only in early events but also in late events in colorectal tumorigenesis. (Cancer Sci 2010)

HOXB9, a gene overexpressed in breast cancer, promotes tumorigenicity and lung metastasis

The mechanisms underlying tumoral secretion of signaling molecules into the microenvironment, which modulates tumor cell fate, angiogenesis, invasion, and metastasis, are not well understood. Aberrant expression of transcription factors, which has been implicated in the tumorigenesis of several types of cancers, may provide a mechanism that induces the expression of growth and angiogenic factors in tumors, leading to their local increase in the tumor microenvironment, favoring tumor progression. In this report, we demonstrate that the transcription factor HOXB9 is overexpressed in breast carcinoma, where elevated expression correlates with high tumor grade. HOXB9 induces the expression of several angiogenic factors (VEGF, bFGF, IL-8, and ANGPTL-2), as well as ErbB (amphiregulin, epiregulin, and neuregulins) and TGF-ß, which activate their respective pathways, leading to increased cell motility and acquisition of mesenchymal phenotypes. In vivo, HOXB9 promotes the formation of large, well-vascularized tumors that metastasize to the lung. Thus, deregulated expression of HOXB9 contributes to breast cancer progression and lung metastasis by inducing several growth factors that alter tumor-specific cell fates and the tumor stromal microenvironment.

Loss of B-cell translocation gene-2 in estrogen receptor-positive breast carcinoma is associated with tumor grade and overexpression of cyclin d1 protein.

The B-cell translocation gene-2 (BTG2) is present in the nuclei of epithelial cells in many tissues, including the mammary gland where its expression is regulated during glandular proliferation and differentiation in pregnancy. In immortalized mammary epithelial cells and breast cancer cells, BTG2 protein localized predominantly to the nucleus and cytoplasm, respectively. The highly conserved domains (BTG boxes A, B, and C) were required for regulating localization, suppression of cyclin D1 and growth inhibitory function of BTG2. Expression analysis of BTG2 protein in human breast carcinoma (n = 148) revealed the loss of nuclear expression in 46% of tumors, whereas it was readily detectable in the nuclei of adjacent normal glands. Loss of nuclear BTG2 expression in estrogen receptor-alpha (ERalpha)-positive breast tumors correlated significantly with increased histologic grade and tumor size. Consistent with its ability to suppress cyclin D1 transcription, loss of nuclear BTG2 expression in ER-positive breast carcinomas showed a significant correlation with cyclin D1 protein overexpression, suggesting that loss of BTG2 may be a factor involved in deregulating cyclin D1 expression in human breast cancer.

Homeobox B9 induces epithelial-to-mesenchymal transition-associated radioresistance by accelerating DNA damage responses

Homeobox 9 (HOXB9), a nontransforming transcription factor overexpressed in breast cancer, alters tumor cell fate and promotes tumor progression and metastasis. Here we show that HOXB9 confers resistance to ionizing radiation by promoting DNA damage response. In nonirradiated cells, HOXB9 induces spontaneous DNA damage, phosphorylated histone 2AX and p53 binding protein 1 foci, and increases baseline ataxia telangiectasia mutated (ATM) phosphorylation. Upon ionizing radiation, ATM is hyperactivated in HOXB9-expressing cells during the early stages of the double-stranded DNA break (DSB) response, accelerating accumulation of phosphorylated histone 2AX, mediator of DNA-damage checkpoint 1, and p53 binding protein 1, at DSBs and enhances DSB repair. The effect of HOXB9 on the response to ionizing radiation requires the baseline ATM activity before irradiation and epithelial-to-mesenchymal transition induced by TGF-β, a HOXB9 transcriptional target. Our results reveal the impact of a HOXB9–TGF-β–ATM axis on checkpoint activation and DNA repair, suggesting that TGF-β may be a key factor that links tumor microenvironment, tumor cell fate, DNA damage response, and radioresistance in a subset of HOXB9-overexpressing breast tumors.

The influence of donor age on liver regeneration and hepatic progenitor cell populations.

BACKGROUND Recent reports suggest that donor age might have a major impact on recipient outcome in adult living donor liver transplantation (LDLT), but the reasons underlying this effect remain unclear. The aims of this study were to compare liver regeneration between young and aged living donors and to evaluate the number of Thy-1+ cells, which have been reported to be human hepatic progenitor cells. METHODS LDLT donors were divided into 2 groups (Group O, donor age ≥ 50 years, n = 6 and Group Y, donor age ≤ 30 years, n = 9). The remnant liver regeneration rates were calculated on the basis of computed tomography volumetry on postoperative days 7 and 30. Liver tissue samples were obtained from donors undergoing routine liver biopsy or patients undergoing partial hepatectomy for metastatic liver tumors. Thy-1+ cells were isolated and counted using immunomagnetic activated cell sorting (MACS) technique. RESULTS Donor liver regeneration rates were significantly higher in young donors compared to old donors (P = .042) on postoperative day 7. Regeneration rates were significantly higher after right lobe resection compared to rates after left lobe resection. The MACS findings showed that the number of Thy-1+ cells in the human liver consistently tended to decline with age. CONCLUSION Our study revealed that liver regeneration is impaired with age after donor hepatectomy, especially after right lobe resection. The declining hepatic progenitor cell population might be one of the reasons for impaired liver regeneration in aged donors.

Breast tumor progression induced by loss of BTG2 expression is inhibited by targeted therapy with the ErbB/HER inhibitor lapatinib

The B-cell translocation gene-2 (BTG2), a p53-inducible gene, is suppressed in mammary epithelial cells during gestation and lactation. In human breast cancer, decreased BTG2 expression correlates with high tumor grade and size, p53 status, blood and lymph vessel invasion, local and metastatic recurrence and decrease in overall survival, suggesting that suppression of BTG2 has a critical role in disease progression. To analyze the role of BTG2 in breast cancer progression, BTG2 expression was knocked down in mammary epithelial cells. Suppression of BTG2 enhances the motility of cells in vitro and tumor growth and metastasis in vivo. The effects of BTG2 knockdown are mediated through stabilization of the human epidermal growth factor receptor (HER) ligands neuregulin and epiregulin and activation of the HER2 and HER3 receptors, leading to elevated AKT phosphorylation. Suppression of HER activation using the tyrosine kinase inhibitor lapatinib abrogates the effects of BTG2 knockdown, including the increased cell migration observed in vitro and the enhancement of tumorigenesis and metastasis in vivo. These results link BTG2-dependent effects on tumor progression to ErbB receptor signaling, and raise the possibility that targeted inhibition of this pathway may be relevant in the treatment of breast cancers that have reduced BTG2 expression.

Cooperation of Cancer Stem Cell Properties and Epithelial-Mesenchymal Transition in the Establishment of Breast Cancer Metastasis

Epithelial-mesenchymal transition (EMT) is a multistep process in which cells acquire molecular alterations such as loss of cell-cell junctions and restructuring of the cytoskeleton. There is an increasing understanding that this process may promote breast cancer progression through promotion of invasive and metastatic tumor growth. Recent observations imply that there may be a cross-talk between EMT and cancer stem cell properties, leading to enhanced tumorigenicity and the capacity to generate heterogeneous tumor cell populations. Here, we review the experimental and clinical evidence for the involvement of EMT in cancer stem cell theory, focusing on the common characteristics of this phenomenon.

Protective effect of high-mobility group box 1 blockade on acute liver failure in rats

High-mobility group box 1 (HMGB1) is a monocyte-derived inflammatory mediator that is released in some conditions including shock, tissue injury, and endotoxin-induced lethality. In this study, we determined the plasma and hepatic tissue levels of HMGB1 in a drug-induced rat acute liver failure (ALF) model and investigated the effect of HMGB1 blockade on ALF. Adult male Sprague-Dawley rats, weighing 250 to 300 g, were used for this study. d-galactosamine was injected into the penile vein to induce ALF. To determine HMGB1 levels, plasma and hepatic tissue samples were serially collected after the d-galactosamine injection. To test the effect of HMGB1 blockade, anti-HMGB1 polyclonal antibodies or control antibodies were injected into the penile vein right after injection of d-galactosamine. Levels of HMGB1 were increased in plasma and decreased in hepatic tissue after induction of ALF. Immunohistochemical examination for HMGB1 showed that liver from animals with ALF had little staining, whereas normal liver had strong staining in the nuclei. Injection of anti-HMGB1 antibodies resulted in significant suppression of plasma HMGB1 and hepatic enzymes, marked suppression of plasma inflammatory cytokines, marked improvement of histological findings, and significant improvement of survival. The decrease of hepatic HMGB1 was also significantly suppressed in the group injected with anti-HMGB1 antibodies. The present study suggests that in ALF, the liver may release HMGB1 into the plasma, and that neutralizing the released HMGB1 has a protective effect against injury.ABBREVIATIONS-ALF-acute liver failure; HMGB1-high-mobility group box 1; ELISA-enzyme-linked immunosorbent assay; anti-HMGB1 Ab-anti-HMGB1 polyclonal antibodies; control Ab-control IgY antibodies; AST-aspartate aminotransferase; ALT-alanine aminotransferase; LDH-lactic dehydrogenase; IL-interleukin; TNF-tumor necrosis factor

The integrin αvβ3-5 ligand MFG-E8 is a p63/p73 target gene in triple negative breast cancers but exhibits suppressive functions in ER+ and erbB2+ breast cancers

The progression from preinvasive lesion to invasive carcinoma is a critical step contributing to breast cancer lethality. We identified downregulation of milk fat globule-EGF factor 8 (MFG-E8) as a contributor to breast cancer progression using microarray analysis of laser capture microdissected (LCM) tissues. We first identified MFG-E8 downregulation in invasive lesions in transgenic mammary tumor models, which were confirmed in LCM-isolated human invasive ductal carcinomas compared with patient-matched normal tissues. In situ analyses of MFG-E8 expression in estrogen receptor (ER) positive cases confirmed its downregulation during breast cancer progression and small inhibitory MFG-E8 RNAs accelerated ER(+) breast cancer cell proliferation. MFG-E8 also decreased in erbB2(+) human cancers and erbB2 transgenic mice lacking MFG-E8 showed accelerated tumor formation. In contrast, MFG-E8 expression was present at high levels in triple-negative (ER(-), PgR(-), erbB2(-)) breast cancers, cell lines, and patient sera. Knockdown, chromatin immunoprecipitation, and reporter assays all showed that p63 regulates MFG-E8 expression, and MFG-E8 knockdowns sensitized triple-negative breast cancers to cisplatin treatment. Taken together, our results show that MFG-E8 is expressed in triple-negative breast cancers as a target gene of the p63 pathway, but may serve a suppressive function in ER(+) and erbB2(+) breast cancers. Its potential use as a serum biomarker that contributes to the pathogenesis of triple-negative breast cancers urges continued evaluation of its differential functions.

Direct inhibition of the transforming growth factor‐β pathway by protein‐bound polysaccharide through inactivation of Smad2 signaling

Transforming growth factor‐β (TGF‐β) is involved in the regulation of cell proliferation, differentiation, and apoptosis and is associated with epithelial–mesenchymal transition (EMT). Inhibition of the TGF‐β pathway is an attractive strategy for the treatment of cancer. We recently screened for novel TGF‐β inhibitors among commercially available drugs and identified protein‐bound polysaccharide (PSK) as a strong inhibitor of the TGF‐β‐induced reporter activity of 3TP‐lux, a TGF‐β1‐responsive luciferase reporter. Protein‐bound polysaccharide is used as a non‐specific immunostimulant for the treatment of gastric and colorectal cancers in Japan. The anticancer activity of this agent may involve direct regulation of growth factor production and enzyme activity in tumors in addition to its immunomodulatory effect. Although several clinical studies have shown the beneficial therapeutic effects of PSK on various types of tumors, its mechanism of action is not clear. In the present study, Western blot analysis showed that PSK suppressed the phosphorylation and nuclear localization of the Smad2 protein, thereby suggesting that PSK inhibits the Smad and MAPK pathways. Quantitative PCR analysis showed that PSK decreased the expression of several TGF‐β pathway target genes. E‐cadherin and vimentin immunohistochemistry showed that PSK suppressed TGF‐β1‐induced EMT, and FACS analysis showed that PSK inhibited the EMT‐mediated generation of CD44+/CD24− cells. These data provide new insights into the mechanisms mediating the TGF‐β‐inhibiting activity of PSK and suggest that PSK can effectively treat diseases associated with TGF‐β signaling. (Cancer Sci 2012; 103: 317–324)