Hirotaka Koizumi

Expression of β-catenin in normal breast tissue and breast carcinoma : a comparative study with epithelial cadherin and α-catenin

Expression of β‐catenin was investigated in normal breast tissue and 66 breast carcinomas in conjunction with expression of epithelial cadherin (E‐CD) and α‐catenin. In normal mammary ducts and acini, intense β‐catenin immunoreactivity was present at the basolateral surfaces of luminal epithelium and weak immunoreactivity was observed at the lateral borders of myoepithelial cells. No β‐catenin was revealed at the myoepithelial basal surface. The intercellular expression of β‐catenin, as well as of E‐CD and α‐catenin, was also observed in carcinoma tissues with varying staining intensity. Almost all of 10 intraductal carcinomas and approximately 70% of 41 invasive ductal carcinomas expressed the three molecules at the same level as in normal glands, whereas approximately 80% of 13 invasive lobular carcinomas showed severe deficiency of them. Two lobular carcinomas in situ showed complete absence of all of the proteins. Some of these findings were confirmed biochemically by immunoblotting analysis. In invasive ductal carcinomas, α‐catenin was reduced more frequently in diffuse than in solid type tumours, whereas the level of expression of β‐catenin and E‐CD was unchanged between them. No correlation was present between reduced expression of the adhesion molecules and lymph node metastasis.

Prediction of breast cancer sensitivity to neoadjuvant chemotherapy based on status of DNA damage repair proteins

IntroductionVarious agents used in breast cancer chemotherapy provoke DNA double-strand breaks (DSBs). DSB repair competence determines the sensitivity of cells to these agents whereby aberrations in the repair machinery leads to apoptosis. Proteins required for this pathway can be detected as nuclear foci at sites of DNA damage when the pathway is intact. Here we investigate whether focus formation of repair proteins can predict chemosensitivity of breast cancer.MethodsCore needle biopsy specimens were obtained from sixty cases of primary breast cancer before and 18-24 hours after the first cycle of neoadjuvant epirubicin plus cyclophosphamide (EC) treatment. Nuclear focus formation of DNA damage repair proteins was immunohistochemically analyzed and compared with tumor response to chemotherapy.ResultsEC treatment induced nuclear foci of γH2AX, conjugated ubiquitin, and Rad51 in a substantial amount of cases. In contrast, BRCA1 foci were observed before treatment in the majority of the cases and only decreased after EC in thirteen cases. The presence of BRCA1-, γH2AX-, or Rad51-foci before treatment or the presence of Rad51-foci after treatment was inversely correlated with tumor response to chemotherapy. DNA damage response (DDR) competence was further evaluated by considering all four repair indicators together. A high DDR score significantly correlated with low tumor response to EC and EC + docetaxel whereas other clinicopathological factors analyzed did not.ConclusionsHigh performing DDR focus formation resulted in tumor resistance to DNA damage-inducing chemotherapy. Our results suggested an importance of evaluation of DDR competence to predict breast cancer chemosensitivity, and merits further studying into its usefulness in exclusion of non-responder patients.

HERC2 Is an E3 Ligase That Targets BRCA1 for Degradation

The breast cancer suppressor BRCA1 forms a stable heterodimeric E3 ubiquitin ligase with BARD1. Each protein controls the abundance and stability of the other, and loss of the interaction leads to BRCA1 degradation. Here, we show that HERC2, a protein recently implicated in DNA damage repair, targets BARD1-uncoupled BRCA1 for degradation. HERC2 shuttles between the nucleus and the cytoplasm. Its COOH-terminal HECT-containing domain interacts with an NH(2)-terminal degron domain in BRCA1. HERC2 ubiquitinates BRCA1; this reaction depends on Cys(4762) of HERC2, the catalytic ubiquitin binding site, and the degron of BRCA1. The HERC2-BRCA1 interaction is maximal during the S phase of the cell cycle and rapidly diminishes as cells enter G(2)-M, inversely correlated with the steady-state level of BRCA1. Significantly, HERC2 depletion antagonizes the effects of BARD1 depletion by restoring BRCA1 expression and G(2)-M checkpoint activity. Conversely, BARD1 protects BRCA1 from HERC2-mediated ubiquitination. Collectively, our findings identify a function for HERC2 in regulating BRCA1 stability in opposition to BARD1. The HERC2 expression in breast epithelial cells and breast carcinomas suggests that this mechanism may play a role in breast carcinogenesis.

Immunohistochemical analysis of TrkA neurotrophin receptor expression in human non-neuronal carcinomas

The Trk family of tyrosine protein kinase receptors plays a significant role in the development and maintenance of neural tissues. It has been recently shown that Trk receptors are also expressed by a wide range of normal non‐neuronal tissues in humans in a cell type‐specific manner. In the present study, the expression patterns of TrkA in 337 non‐neuronal invasive carcinomas of 15 different human tissues were investigated immunohistochemically. Overall, 133 (39%), 101 (30%) and 103 (31%) tumors exhibited strong, moderate and no TrkA Immunoreactivity, respectively. Esophageal and thyroid carcinomas expressed high levels of TrkA, whereas the levels in gastric and colon cancers were low. TrkA expression was detected not only in carcinomas originating from TrkA‐positive normal counterpart tissues, Including the esophagus, breast, lung and uterus, but also in those from TrkA‐negative tissues/cells of the thyroid, liver and ovary. Immunostaining for nerve growth factor‐β, the specific ligand for TrkA, in esophageal and breast carcinomas demonstrated its immunoreactivity in stromal fibroblasts and some TrkA‐expressing tumor cells. These results suggest that paracrine/autocrine regulation via stromal/tumoral NGF‐tumoral TrkA interaction may be involved In the growth of certain non‐neuronal carcinomas.

Involvement of cell‐mediated killing in apoptosis in histiocytic necrotizing lymphadenitis (Kikuchi‐Fujimoto disease)

Histiocytic necrotizing lymphadenitis, also called Kikuchi‐Fujimoto (KF) disease, is a benign disorder characterized histologically by paracortical necrotic foci surrounded by histiocytic aggregates. We analysed affected lymph node tissues from 34 patients with the disease in an attempt to elucidate its histogenesis. The ‘necrotizing’ cells showed typical apoptotic changes, including cell shrinkage and condensed and fragmented nuclei. Apoptotic bodies with a peculiar ultrastructure were demonstrated, and DNA fragmentation was detected in these cells by in situ end labelling. Immunostaining for the apoptosis‐regulating proteins bcl‐2, bax, c‐myc and p53 failed to show their involvement in KF disease. However, perforin, a killer cell‐specific cytolytic protein essential for provoking apoptosis in target cells, was found to be expressed abundantly by the infiltrating cells, which were thought to be cytotoxic T‐lymphocytes. Perforin‐expressing cells were present in the apoptotic foci of 28 of the 34 patients (82.4%). Virtually no cells containing perforin granules were present in non‐pathological regions, lymph node tissues from control subjects with reactive or tuberculous lymphadenitis or those from patients with KF disease with negligible apoptosis. Therefore, the ‘necrosis’ associated with KF disease appears to be attributable to trans apoptotic death of the killer cell target in the affected nodes. We propose that KF disease should be called apoptotic lymphadenitis.

Overexpression of heat shock protein 27 in squamous cell carcinoma of the uterine cervix: a proteomic analysis using archival formalin-fixed, paraffin-embedded tissues.

Proteomic analysis of squamous cell carcinoma of the uterine cervix was performed using total protein from archival formalin-fixed, paraffin-embedded tissues. A wide range of proteins with molecular weights of 10 to greater than 200 kd was extracted from formalin-fixed, paraffin-embedded tissues using a recently developed protocol based on the heat-induced antigen retrieval technique. The extracted proteins from normal squamous epithelium (n = 53) and squamous cell carcinoma (n = 21) were fluorescently labeled and separated using 2-dimensional gel electrophoresis. We identified 728 differentially expressed proteins, with 144 up-regulated and 584 down-regulated as compared with normal squamous epithelial tissue samples. Nine proteins showing pronounced up-regulation in squamous cell carcinoma were analyzed on liquid chromatography-tandem mass spectrometry. Among the candidate proteins identified, minichromosome maintenance 8, a disintegrin and metalloproteinase domain 18, and heat shock protein 27 were analyzed in Western blotting, resulting in significant overexpression of heat shock protein 27 in squamous cell carcinoma over normal mucosa (P < .05). Furthermore, immunostaining revealed heat shock protein 27 overexpression not only in squamous cell carcinoma but in various stages of cervical intraepithelial neoplasia (grades 1-3, n = 90), including dysplasia and carcinoma in situ. The expression levels of heat shock protein 27 in cervical intraepithelial neoplasia grades 1 to 3 and squamous cell carcinoma were significantly higher than that in normal mucosa (P < .05). In the neoplastic lesions, heat shock protein 27 expression levels in cervical intraepithelial neoplasia grade 3 and squamous cell carcinoma were significantly higher than that in cervical intraepithelial neoplasia grade 1 (P < .05). These results may suggest a role of heat shock protein 27 in tumor development and progression in the cervical intraepithelial neoplasia-squamous cell carcinoma sequence. Future experiments using formalin-fixed, paraffin-embedded tissue-based proteomic analysis will be a powerful tool for various pathologic studies.

In vitro ubiquitination of cyclin D1 by ROC1–CUL1 and ROC1–CUL3

Overexpression of cyclin D1 has been implicated in a variety of tumors, such as breast cancers, gastrointestinal cancers and lymphomas. Both gene amplification and protein degradation mediated by ubiquitin (Ub)‐dependent proteolysis regulate the abundance of cyclin D1. Here we report that ROC1 interacted with all three D type cyclins in vivo but did not bind to other cyclins tested. The ROC1–CUL1 and ROC1–CUL3, but not ROC1–CUL2, –CUL3 and –CUL4, immunocomplexes promoted polyubiquitination of bacterially purified cyclin D1 in vitro. RING finger mutations of ROC1 eliminated the Ub ligase activity toward cyclin D1. In all cases the ubiquitination of cyclin D1 was accompanied by autoubiquitination of the cullins. The results suggest the involvement of ROC1–cullin ligases in cyclin D1 ubiquitination and a potential mechanism whereby the cullin subunit is ubiquitinated itself while ubiquitinating a substrate.

Close association of IASLC/ATS/ERS lung adenocarcinoma subtypes with glucose-uptake in positron emission tomography

OBJECTIVES Correlations between maximum standardized uptake value (SUVmax) in fluorodeoxyglucose positron emission tomography (FDG-PET) and IASLC/ATS/ERS histopathologic subtypes of lung adenocarcinoma remain unclear. Therefore, the aim of this study was to retrospectively clarify associations between SUVmax and adenocarcinoma subtypes with postoperative outcomes. MATERIALS AND METHODS Associations of SUVmax measured in preoperative FDG-PET/CT and histologic subtypes of lung adenocarcinoma resected in our hospital were analyzed retrospectively. Overall and disease-free survival rates after surgery were calculated by the Kaplan-Meier method, and survival differences between patient groups were tested by the log-rank test. Multivariate analysis for survival was performed using the Cox regression model. RESULTS A total of 255 patients (130 men and 125 women; mean age, 69 years; range, 22-88 years) were included in the study. Clinical stages included IA in 151 patients, IB in 79, IIA in 9, IIB in 10, and IIIA in 6. SUVmax was closely associated with histologic subtype in resected specimens (p<0.0001). Values were highest in micropapillary predominant invasive adenocarcinoma (MPA) followed by solid predominant (SPA), invasive mucinous (IMA), acinar predominant (APA), papillary predominant (PPA), lepidic predominant (LPA), minimally invasive adenocarcinoma (MIA), and adenocarcinoma in situ (AIS). When the subtypes were classified into three subgroups [group A, AIS+MIA+LPA (low risk); group B, APA+PPA+IMA (intermediate risk); and group C, SPA+MPP (high risk)] by expected postoperative prognoses, there were significant differences in SUVmax among the subgroups corresponding to recurrence risk (p=0.0001). CONCLUSION Preoperative SUVmax was closely associated with both adenocarcinoma subtype and aggregated subgroups, reflecting malignant grade of the tumor and prognosis.

Proteomic analysis of differential protein expression by brain metastases of gynecological malignancies

Brain metastases of gynecological malignancies are rare, but the incidence is increasing. Patients with brain metastases have a poor prognosis, therefore early detection and optimal management is necessary. In order to determine a new biomarker, we aimed to identify proteins that associated with brain metastases. We investigated proteins associated with brain metastases of gynecological malignancies in three patients who underwent surgical resection (stage IIb cervical cancer, stage Ib endometrial cancer, and stage IIIb ovarian cancer). Proteomic analysis was performed on formalin-fixed paraffin-embedded (FFPE) samples of the primary tumors and brain metastases, which were analyzed by liquid chromatography with tandem mass spectrometry. Thereafter, candidate proteins were identified by the Scaffold system and Mascot search program, and were analyzed using western blotting and immunohistochemistry. As a result, a total of 129 proteins were identified. In endometrial and ovarian cancers, western blotting revealed that the expression of alpha-enolase (ENO1) and triosephosphate isomerase (TPI-1) was higher and the expression of Transgelin-2 (TAGLN2) was lower in metastatic tumors than in primary tumors. On the other hand, the expression of TPI-1 and TAGLN2 was lower in metastatic tumors than in primary tumors in cervical cancer. Immunohistochemistry confirmed that ENO1 expression was elevated in the metastatic tumors compared with the primary tumors. In conclusion, the present study showed that FFPE tissue-based proteomics analysis can be powerful tool, and these findings suggested that ENO1, TPI-1, and TAGLN2 may have a role in the development and progression of brain metastasis from gynecological malignancies.

Demonstration of apoptosis in neuroblastoma and its relationship to tumour regression.

The in vivo occurrence of apoptosis in neuroblastomas was investigated. Histologically, a number of tumour cells showed typical apoptotic changes, including cell shrinkage, condensed and fragmented nuclei, eosinophilic cytoplasm, and absence of the inflammatory response. These cells coincided closely with the so-called karyorrhectic cells. An electrophoretic DNA ladder, a functional hallmark of apoptosis, was demonstrated in four of six tumours, and DNA fragmentation was detected in situ by terminal deoxytransferase-mediated nick end-labelling in 26 of 35 tumour specimens (74%). The labelled cell counts ranged from 5 to 62 per 5000 tumour cells (mean±SD: 15.0±14.5). Immunoperoxidase staining revealed that an apoptosis-suppressing protein, bcl-2, was expressed abundantly in advanced-stage tumours, whereas it was absent from karyorrhectic-apoptotic cells. Several tumours with the potential for spontaneous regression were bcl-2-deficient. Immunostaining of the Fas receptor for apoptosis demonstrated that the tumour cells expressed this molecule on their cell surfaces. Our results provide evidence of apoptosis in neuroblastomas and suggest that bcl-2 and the Fas receptor may play a role in its regulatory mechanisms.