Yasuhito Kobayashi

Aryl hydrocarbon receptor suppresses intestinal carcinogenesis in ApcMin/+ mice with natural ligands

Intestinal cancer is one of the most common human cancers. Aberrant activation of the canonical Wnt signaling cascade, for example, caused by adenomatous polyposis coli (APC) gene mutations, leads to increased stabilization and accumulation of β-catenin, resulting in initiation of intestinal carcinogenesis. The aryl hydrocarbon receptor (AhR) has dual roles in regulating intracellular protein levels both as a ligand-activated transcription factor and as a ligand-dependent E3 ubiquitin ligase. Here, we show that the AhR E3 ubiquitin ligase has a role in suppression of intestinal carcinogenesis by a previously undescribed ligand-dependent β-catenin degradation pathway that is independent of and parallel to the APC system. This function of AhR is activated by both xenobiotics and natural AhR ligands, such as indole derivatives that are converted from dietary tryptophan and glucosinolates by intestinal microbes, and suppresses intestinal tumor development in ApcMin/+ mice. These findings suggest that chemoprevention with naturally-occurring and chemically-designed AhR ligands can be used to successfully prevent intestinal cancers.

The ubiquitin ligase CHIP acts as an upstream regulator of oncogenic pathways.

CHIP is a U-box-type ubiquitin ligase that induces ubiquitylation and degradation of its substrates, which include several oncogenic proteins. The relationship between CHIP and tumour progression, however, has not been elucidated. Here, we show that CHIP suppresses tumour progression in human breast cancer by inhibiting oncogenic pathways. CHIP levels were negatively correlated with the malignancy of human breast tumour tissues. In a nude mouse xenograft model, tumour growth and metastasis were significantly inhibited by CHIP expression. In contrast, knockdown of CHIP (shCHIP) in breast cancer cells resulted in rapid tumour growth and metastastic phenotypes in mice. In cell-based experiments, anchorage-independent growth and invasiveness of shCHIP cells was significantly elevated due to increased expression of Bcl2, Akt1, Smad and Twist. Proteomic analysis identified the transcriptional co-activator SRC-3 (refs 13, 14, 15, 16, 17, 18, 19) as a direct target for ubiquitylation and degradation by CHIP. Knocking down SRC-3 in shCHIP cells reduced the expression of Smad and Twist, and suppressed tumour metastasis in vivo. Conversely, SRC-3 co-expression prevented CHIP-induced suppression of metastasis formation. These observations demonstrate that CHIP inhibits anchorage-independent cell growth and metastatic potential by degrading oncogenic proteins including SRC-3.

Estrogen receptor β is expressed in human stomach adenocarcinoma

AbstractPurpose. In stomach adenocarcinoma, the role of the hormonal receptor, estrogen receptor (ER), has been controversial. Recently, a new estrogen receptor, called estrogen receptor β (ERβ), was found to be expressed in various tissues including normal gastrointestinal tract. In this paper, the expression of ERβ in stomach adenocarcinomas has been investigated for the first time, specifically in signet ring cell adenocarcinomas, together with surrounding non-cancerous tissues. Methods. By immunohistochemistry the expression of ERα and β was studied in 29 stomach adenocarcinomas, ten signet ring cell adenocarcinomas, and 19 other adenocarcinomas. Western blotting was performed to examine the immunohistochemical result. Statistical studies (Students t test and χ2-test) explored the relation between the immunohistochemical result and clinicopathological characteristics. Results. All 29 adenocarcinomas, including the signet ring cell ones, demonstrated clear ERβ nucleus staining. Lymphocytes, venous endothelial cells, smooth muscle, and non-cancerous stomach glands also showed strong ERβ staining, while no staining was observed in the immunohistochemistry of ERα. Western blotting showed equivalent ERβ protein levels in cancerous and non-cancerous tissues, which was consistent with the results of immunohistochemical staining. Among signet ring cell adenocarcinomas of the stomach, cytoplasm were stained in addition to nuclei, specifically in patients under the age of 40 years. Conclusions. Our results imply that the effects of estrogen in stomach cancer, as well as those in normal stomach, may be mediated by ERβ, and that the role of ERβ may differ by the subtype of stomach adenocarcinoma – specifically signet ring cell adenocarcinomas and other ones – although large scale samples are needed to confirm these findings.

Expression of sex steroid hormone receptors in human cornea

Purpose. Previously we reported the occurrence of estrogen receptor a (ERa), estrogen receptor ß (ERß) and androgen receptor (AR) in mouse corneas. The present study was designed to investigate the occurrence of various sex steroid hormone receptors, including ERa, progesterone receptor (PR) and AR, in human corneas. Methods. We used reverse transcription-polymerase chain reaction (RT-PCR) to look for sex hormone receptor mRNAs (ERa, PR and AR) in human corneal epithelial cells obtained at autopsy. Next, using an immunocytochemical technique, we localized these receptors in donor human corneas. Results. mRNAs encoding all receptors tested for were found in corneal epithelial cells obtained from male and female donor eyes. Immunocytochemical examination revealed that the receptors were located in the nuclei of corneal epithelial, stromal, and endothelial cells. Conclusions. Since receptors for both male and female sex hormones are present in human corneas of both genders, we postulate that the receptors may influence the biological functions of corneal cells through direct interaction with specific hormones.

Prediction of prognosis of estrogen receptor‐positive breast cancer with combination of selected estrogen‐regulated genes

Estrogen receptor (ER)‐positive breast cancer is a distinct subpopulation of breast cancer exhibiting a high response rate to endocrine therapy. However, not all ER‐positive patients respond to the therapy, and a subgrouping of ER‐positive patients based on the physiology of estrogen signaling is expected to be useful for predicting the prognosis. This study has revealed that selected estrogen‐regulated genes (ERGs) are useful in identification of a poor‐prognosis population among ER‐positive breast cancer patients. First, the expression levels of 11 ERGs, selected based on our earlier microarray study in cultured cells, were analyzed by means of real‐time reverse transcription‐PCR in 14 ER‐positive human breast cancer tissues. The patients were clearly divided into two groups in cluster analysis. Then, we examined the expression levels of two representative ERGs, histone deacetylase 6 (HDAC6) and insulin‐like growth factor binding protein 4 (IGFBP‐4), In 62 ER‐positive patients with immunohistochemistry to assess the impact of ERG expression on prognosis (median follow‐up 4409 days). Positive HDAC6 staining was significantly correlated with a lower disease‐free survival rate. Moreover, when the expression level of HDAC6 was assessed in combination with IGFBP‐4 expression in the nucleus, the poor‐prognosis patients were more accurately identified. This study has identified new candidate ERGs for prediction of prognosis, and we suggest that combined assessment of the expression levels of these ERGs will contribute to the clinically useful stratification of ER‐positive breast cancer patients.

Tumor-Stromal Interaction through the Estrogen-Signaling Pathway in Human Breast Cancer

In postmenopausal breast cancers, locally produced estrogen by adipose stromal cells causes the progression of tumor growth. Although aromatase, a key enzyme of estrogen synthesis, is highly expressed in the adipose stromal cells, and aromatase inhibitors show greater efficacy in postmenopausal breast cancers, the mechanism of increasing aromatase activity in the stromal cells remains unclear. To analyze the estrogen signals and to detect the estrogen receptor (ER)-activating ability of adipose stromal cells for individual human breast cancers, we developed a new reporter cell system. To visualize the activation of ER, we first established a stable transformant, named E10, of human breast cancer MCF-7 cells by transfection with the estrogen-responsive element-green fluorescent protein (GFP) gene. E10 cells specifically express GFP when ER is activated by estrogen or by coculture with adipose stromal cells isolated from breast tumor tissues in the presence of testosterone, a substrate for aromatase. Treatment of adipose stromal cells with dexamethasone, a stimulator of aromatase gene expression, resulted in an increase in the expression of GFP in E10 cells in the coculture. Using this system, we characterized the adipose stromal cells of 67 human breast cancers and found that GFP expression levels vary among the cases, suggesting that the ability of adipose stromal cells to activate ERs is unique for individual breast cancers. High induction levels of GFP were observed more frequently in postmenopausal cases than in premenopausal cases, whereas they did not significantly correlate with the ER expression status. Aromatase inhibitors inhibited the induction of GFP expression in the coculture, but the sensitivities to the drugs varied among the individual cases. Aromatase gene expression levels in adipose stromal cells did not always correlate with their ability to induce GFP. These results suggest that this system to detect total ER activation based on the interaction with adipose stromal cells is a useful tool for analyzing local estrogen signals and for tumor-stromal interactions.

Evaluation of aspartic acid racemization ratios in the human femur for age estimation.

Levels of D-aspartic acid (D/L ratio) in cranial non-collagen proteins (acid-soluble peptide fractions) have been reported to increase with age. We isolated total amino acid fractions from the femur and separately isolated acid-insoluble collagen fraction and acid-soluble peptide fractions; then D/L ratios were measured from each fraction by gas chromatography. We evaluated the applicability of their D/L ratios for age estimation based on their correlation coefficient. A sex-related difference was observed in the D/L ratio. In particular, aged females showed a low ratio, suggesting an association with bone disorders. In males, the D/L ratios of acid-soluble peptide fraction showed the highest correlation rate (r = 0.969) with age, and those of total amino acid fraction showed the highest correlation rate (r = 0.633) with age in females. Without separation of male and female, the D/L ratios of total amino acid fraction showed the highest value (r = 0.853). The D/L ratio of acid-soluble peptide fractions differed according to the size of bone powder particles, being higher for larger particle sizes. These results suggest that the application of D/L ratio from total amino acid fraction is the most effective method for estimating age using the human femur. However, care is necessary when studing cadavers that might be females.

Molecular markers for reinforcement of histological subclassification of neuroendocrine lung tumors

The degree of malignancy of neuroendocrine lung tumors (NEs) increases in this order: from typical carcinoids (TCs) through atypical carcinoids (ACs) to large cell neuroendocrine carcinomas (LCNECs) and small cell lung carcinomas (SCLCs). However, histological classification has sometimes proved difficult. We here investigated loss of heterozygosity (LOH) using eight microsatellite markers and expression of p53, Bcl‐2 and Bax proteins using immunohistochemical methods in 57 NEs (19 TCs, 5 ACs, 14 LCNECs and 19 SCLCs), looking for objective genetic markers to distinguish between subtypes. The frequencies of LOHs on D3S1300, RBi2 and TP53, the combinations of LOH status for RBi2 and TP53, and the immunohistochemically demonstrated Bcl‐2/Bax ratios and p53‐positive rates significantly differed among histopathologically diagnosed NEs. Differentiation between TC and AC was possible with reference to LOH on D3S1300, RBi2 and TP53, and the combined LOH status on RBi2 and TP53 (i.e., both LOH(‐) versus one LOH(+)). For comparison between AC and LCNEC+SCLC, LOH on TP53 or the combination of two markers—one LOH(+) versus both LOH(+)—was applied. Furthermore, in three discordant cases of diagnoses based on histology and LOH markers, diagnoses using the latter were considered to be more probable by survival analysis. The present study indicated that assessment of LOHs using microsatellite markers could provide objective markers that can distinguish subtypes of NEs, for which histological assessment may commonly result in disagreement.

A mouse model of Waardenburg syndrome type 4 with a new spontaneous mutation of the endothelin-B receptor gene

Waardenburg syndrome (WS) is a hereditary auditory-pigmentary syndrome with hearing impairment and pigmentation anomaly of the skin and iris. In addition to these major symptoms, WS type 4 is associated with Hirschsprung disease. To date, three genes responsible for WS4 have been cloned: genes for a transcription factor SOX10, endothelin 3 (EDN3), and endothelin B receptor (EDNRB). We here describe a novel mutant mouse with a mutation of the Ednrb gene, and propose the mouse as an animal model of WS4. These mutants are with mixed genetic background of BALB/c and MSM (an inbred strain of Japanese wild mice) and have extensive white spotting. They died between 2 and 7 weeks after birth owing to megacolon: their colon distal to the megacolon lacked Auerbach’s plexus cells. Interestingly, these mutants did not respond to sound, and the stria vascularis of their cochlea lacked intermediate cells, i.e., neural crest-derived melanocytes. Since these symptoms resembled those of human WS4 and were transmitted in autosomal recessive hereditary manner, the mutants were named WS4 mice. Breeding analysis revealed that WS4 mice are allelic with piebald-lethal and JF1 mice, which are also mutated in the Ednrb gene. Mutation analysis revealed that their Ednrb lacked 318 nucleotides encoding Ednrb transmembrane domains owing to deletion of exons 2 and 3. Interaction between endothelin 3 and its receptor is required for normal differentiation and development of melanocytes and Auerbach’s plexus cells. We concluded that a missing interaction here led to a lack of these cells, which caused pigmentation anomaly, deafness, and megacolon in WS4 mice.

Different Subtypes of Human Lung Adenocarcinoma Caused by Different Etiological Factors : Evidence from p53 Mutational Spectra

Human lung adenocarcinomas are only relatively weakly associated with tobacco smoke, and other etiological factors need to be clarified. These may also vary with the histopathology. Because the p53 mutation status (frequency and spectrum) of a carcinoma can provide clues to causative agents, we subclassified 113 adenocarcinomas into five cell types: hobnail, columnar/cuboidal, mixed, polygonal, and goblet (54, 23, 18, 13, and 5, respectively) and investigated relationships with p53 mutations and smoking history. In the hobnail cell type, a low mutational frequency (37%) and a high proportion of transitions (65%), especially G:C to A:T transitions at CpG dinucleotides (45%) associated with spontaneous mutations, were found with a weak relation to tobacco smoke. In contrast, a high mutation frequency (70%) with a higher proportion of transversions (50%), especially G:C to T:A (44%) on the nontranscribed DNA strand, caused by exogenous carcinogenic agents like tobacco smoke, were observed for the columnar cell type, as with squamous cell carcinomas. These results indicate that two major subtypes of lung adenocarcinoma exist, one probably caused by tobacco smoke, and the other possibly due to spontaneous mutations. For the prevention of lung adenocarcinomas, in addition to stopping tobacco smoking, the elucidation of endogenous mechanisms is important.