Masafumi Kurosumi

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.

Development of automated brightfield double In Situ hybridization (BDISH) application for HER2 gene and chromosome 17 centromere (CEN 17) for breast carcinomas and an assay performance comparison to manual dual color HER2 fluorescence In Situ hybridization (FISH)

BackgroundHuman epidermal growth factor receptor 2 (HER2) fluorescence in situ hybridization (FISH) is a quantitative assay for selecting breast cancer patients for trastuzumab therapy. However, current HER2 FISH procedures are labor intensive, manual methods that require skilled technologists and specialized fluorescence microscopy. Furthermore, FISH slides cannot be archived for long term storage and review. Our objective was to develop an automated brightfield double in situ hybridization (BDISH) application for HER2 gene and chromosome 17 centromere (CEN 17) and test the assay performance with dual color HER2 FISH evaluated breast carcinomas.MethodsThe BDISH assay was developed with the nick translated dinitrophenyl (DNP)-labeled HER2 DNA probe and DNP-labeled CEN 17 oligoprobe on the Ventana BenchMark® XT slide processing system. Detection of HER2 and CEN 17 signals was accomplished with the silver acetate, hydroquinone, and H2O2 reaction with horseradish peroxidase (HRP) and the fast red and naphthol phosphate reaction with alkaline phosphatise (AP), respectively. The BDISH specificity was optimized with formalin-fixed, paraffin-embedded xenograft tumors, MCF7 (non-amplified HER2 gene) and BT-474 (amplified HER2 gene). Then, the BDISH performance was evaluated with 94 routinely processed breast cancer tissues. Interpretation of HER2 and CEN 17 BDISH slides was conducted by 4 observers using a conventional brightfield microscope without oil immersion objectives.ResultsSequential hybridization and signal detection for HER2 and CEN 17 ISH demonstrated both DNA targets in the same cells. HER2 signals were visualized as discrete black metallic silver dots while CEN 17 signals were detected as slightly larger red dots. Our study demonstrated a high consensus concordance between HER2 FISH and BDISH results of clinical breast carcinoma cases based on the historical scoring method (98.9%, Simple Kappa = 0.9736, 95% CI = 0.9222 – 1.0000) and the ASCO/CAP scoring method with the FISH equivocal cases (95.7%, Simple Kappa = 0.8993%, 95% CI = 0.8068 – 0.9919) and without the FISH equivocal cases (100%, Simple Kappa = 1.0000%, 95% CI = 1.0000 – 1.0000).ConclusionAutomated BDISH applications for HER2 and CEN 17 targets were successfully developed and it might be able to replace manual two-color HER2 FISH methods. The application also has the potential to be used for other gene targets. The use of BDISH technology allows the simultaneous analyses of two DNA targets within the context of tissue morphological observation.

Detection of HER-2/neu (c-erb B-2) DNA amplification in primary breast carcinoma

Fluorescent in situ hybridization (FISH) has been shown to be one of the most reliable methods with which to estimate the status of the HER‐2/neu (or c‐erb B‐2) oncogene at the DNA level.

Histopathological criteria for assessment of therapeutic response in breast cancer (2007 version)

In preoperative drug therapy (chemo-, endocrine, molecular-targeting, and other therapies) and/or radiotherapy for breast cancer, various degrees of histological changes of cancer tissue occur depending on the sensitivity of cancer cells to the particular treatment, kinds of therapeutic agents, dosage of drugs, method of administration, kind of combination, type and dosage of isotope, method of irradiation, duration of treatment, interval from the last treatment, and resection of cancer tissue. Histopathological criteria for assessment of response to neoadjuvant therapy for breast cancer have been established according to the degree of histological change of cancer tissue as described below. Histological diagnosis should be confirmed before treatment using biopsy materials, and it is important to compare the histopathological findings of cancer tissues before and after treatment for the assessment of response to therapy. Pathological response should be evaluated only from histological changes in the invasive area, and the presence of residual ductal components should be described. If only ductal components remain, the pathological response is evaluated as Grade 3.

Anti-HER2 antibody enhances the growth inhibitory effect of anti-oestrogen on breast cancer cells expressing both oestrogen receptors and HER2

Anti-oestrogen is effective for the treatment of oestrogen receptor (ER)-positive breast carcinomas, butmost of these tumours become resistant to anti-oestrogen. It has been suggested that anti-oestrogen therapy may induce a HER2signalling pathway in breast cancer cells and this may cause resistance to anti-oestrogen. Thus, it is conceivable that combinedtherapy with anti-oestrogen and anti-HER2 antibody might be more effective. In the present study, we investigated the effect ofcombined treatment with a humanized anti-HER2 monoclonal antibody, rhumAbHER2 (trastuzumab), and an anti-oestrogen, ICI 182,780, onthe cell growth of three human breast cancer cell lines which respectively express different levels of ER and HER2. The combinedtreatment enhanced the growth inhibitory effect on ML-20 cells, which express a high level of ER and a moderate level of HER2, butshowed no additive effect on either KPL-4 cells, which express no ER and a moderate level of HER2, or MDA-MB-231 cells, which express no ER and a low level of HER2. It is also suggested that both the antibody and anti-oestrogen induce a G1–S blockade and apoptosis. These findings indicate that combined treatment with anti-HER2 antibody and anti-oestrogen may be useful for thetreatment of patients with breast cancer expressing both ER and HER2.

Clinicopathological analyses of triple negative breast cancer using surveillance data from the Registration Committee of the Japanese Breast Cancer Society

BackgroundTriple negative (TN) breast cancer is defined as a subtype that is negative for estrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor 2 (HER2). To clarify the characteristics of TN breast cancer, surveillance data of the Registration Committee of the Japanese Breast Cancer Society were analyzed.MethodOf 14,748 cases registered in 2004, 11,705 (79.4%) were examined for ER, PgR, and HER2. Of these, the most prevalent (53.8%) was a hormone-responsive subtype with ER positive/PgR positive/HER2 negative, followed by TN subtype (15.5%).ResultsThe proportion of postmenopausal patients was relatively high in the TN subtype. This cancer was diagnosed at a slightly advanced stage and with more cases positive for lymph node metastases than other subtypes. Morphologically, the TN subtype was more frequently classified as solid-tubular carcinoma. Mucinous, tubular, or secretary carcinomas were frequently found in the hormone receptor positive/HER2 negative subtype, while squamous cell carcinoma, spindle cell carcinoma, and metaplastic carcinoma with bone/cartilage metaplasia were very frequently found in the TN group. Apocrine carcinoma was also found very frequently in the TN group. Selection of chemotherapy was not based on receptor subtypes, but was determined by the degree of tumor progression.ConclusionsAlthough TN types are similar to basal-like breast tumor, as determined by gene profiling, their diagnosis needs verification by determination of the level of epidermal growth factor receptor or cytokeratin 5/6 expression. TN type should be examined further for immunohistochemical features and analyzed for prognostic details in this cohort.

A Radicicol Derivative, KF58333, Inhibits Expression of Hypoxia-inducible Factor-1α and Vascular Endothelial Growth Factor, Angiogenesis and Growth of Human Breast Cancer Xenografts

A novel oxime derivative of radicicol, KF58333, binds to the heat shock protein 90 (Hsp90) and destabilizes its associated signaling molecules. These effects play a critical role in the growth inhibition of tumor cells. To further investigate the effects of this agent, it was administered to two human breast cancer cell lines, KPL‐1 and KPL‐4, both in vitro and in vivo. KF58333 dose‐dependently inhibited the growth and vascular endothelial growth factor (VEGF) secretion, concomitantly with a decrease in VEGF mRNA expression, in each cell line. This agent also suppressed the increase of VEGF secretion and expression induced by hypoxia (1% O2). Intravenous injections of this agent into nude mice bearing either KPL‐1 or KPL‐4 xenografts significantly inhibited the tumor growth associated with a decrease in the Ki67 labeling index and microvascular area and an increase in apoptosis and the necrotic area. These findings indicate that the antitumor activity of this radicicol derivative may be partly mediated by decreasing VEGF secretion from tumor cells and inhibiting tumor angiogenesis. To explore the action mechanisms of the anti‐angiogenic effect, the expression level of hypoxia‐inducible factor (HIF)‐lα was investigated. KF58333 provided a significant decrease in the HTF‐lα protein expression under both normoxic and hypoxic conditions. In contrast, the mRNA expression of HIF‐lα was not decreased by this agent. It is suggested that the post‐transcriptional down‐regulation of HIF‐lα expression by this agent may result in a decrease of VEGF expression and tumor angiogenesis.

A new human breast cancer cell line, KPL-1 secretes tumour-associated antigens and grows rapidly in female athymic nude mice.

We recently established a new human breast cell line, designated KPL-1, which was derived from the malignant effusion of a patient with breast cancer. This cell line is highly tumorigenic and grows rapidly in female nude mice. Cytogenetic analysis indicated its human origin and revealed a hypertriploid modal number of chromosomes. Electron microscopic examination suggested that the KPL-1 cells are of epithelial origin. Immunohistochemical studies revealed that the cells express cytokeratin, carcinoembryonic antigen and CA 15-3. They also possess a large number of oestrogen receptors but not progesterone receptors. Interestingly, KPL-1 cells seem to grow oestrogen independently in vitro. No amplification of c-erbB-2, c-myc, H-ras and N-ras genes was detected. KPL-1 cells secrete a large amount of tissue polypeptide antigen (TPA). Although the secretion of CA 15-3 seemed to be constant throughout all cell growth phases, TPA secretion increased during the exponential growth phase and decreased during the plateau phase. Serum TPA levels significantly correlated with the volume of KPL-1 tumours transplanted into nude mice. These data suggest that this KPL-1 cell line may be useful for studying oestrogen-independent growth and the kinetics of tumour-associated antigens in vivo as well as in vitro.

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.