[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: Sessile serrated adenoma/polyps (SSA/Ps) of the colon are thought to be precursors of sporadic carcinomas. Although it is suggested that SSA/P may grow rapidly from the early stage, its cell kinetics remains obscure. To solve this problem, we analyzed the mitotic and apoptotic activity of normal crypts, microvesicular hyperplastic polyps (MVHPs), and tubular adenomas (TAs), using phospho-histone H3 and cleaved caspase 3 immunohistochemistry. The mitotic index for SSA/Ps (mean, 5.63) and TAs (6.98) was significantly higher than those for normal crypts (2.72) and MVHPs (2.86). Of all tested lesions, the apoptotic index was lowest for SSA/Ps (0.96; normal, 2.71; MVHPs, 2.62; TAs, 6.01) with statistically significant differences. The net growth ratio was close to 1.0 in normal crypts (1.07) while remaining low in MVHPs (1.06) and TAs (1.38), but was markedly elevated in SSA/Ps (7.32, P < 0.01) due to the large imbalance between mitosis and apoptosis. As to apoptosis regulatory proteins, expression of anti-apoptotic Bcl-2 was significantly reduced or undetectable in MVHPs and SSA/Ps, while TAs showed stronger staining than normal crypts. Expression of pro-apoptotic Bax and its activators, Bim and Bad, was significantly reduced in MVHPs and SSA/Ps. We suggest that other complex mechanisms may act synergistically with Bax, Bim, or Bad deficiency to regulate apoptosis suppression in SSA/Ps.
Archiv für Pathologische Anatomie und Physiologie und für Klinische Medicin 01/2013; · 2.68 Impact Factor
[show abstract][hide abstract] ABSTRACT: Malignant mesothelioma (MM) is a rare disease with a poor prognosis. Pleural mesothelioma, which is the most common type of MM, is considered to be caused by asbestos exposure and is increasing in incidence, with about 15,000 new cases diagnosed worldwide annually. On the other hand, peritoneal mesothelioma is a very rare type of MM; thus, its pathogenesis is even less understood than pleural mesothelioma. Recent research on the pathogenesis of malignant pleural mesothelioma has indicated that both epigenetic and genetic alterations contribute to tumorigenesis. Here, we hypothesize that peritoneal mesothelioma also has an epigenetic alteration in the same genes (Kazal-type serine peptidase inhibitor domain 1 (KAZALD1), transmembrane protein 30B (TMEM30B), and mitogen-activated protein kinase 13 (MAPK13)). Our goal is to identify DNA methylation of these three candidate genes in two peritoneal mesothelioma cases. Laser capture microdissection was used to separate diseased sections of formalin-fixed paraffin-embedded samples from one surgically resected tissue (epithelial type) and one autopsy tissue (sarcomatous type). Genomic DNA was subsequently extracted by the standard phenol chloroform method. The DNA was then treated with sodium bisulphite, and pyrosequencing analysis was used to quantitatively analyze the methylation of candidate genes reported to be hypermethylated in malignant pleural mesothelioma (KAZALD1, TMEM30B, and MAPK13). TMEM30B and MAPK13 were not methylated in either case. However, KAZALD1 was highly methylated in sarcomatoid-type peritoneal mesothelioma. We first report that the KAZALD1 gene was hypermethylated in sarcomatoid-type malignant peritoneal mesothelioma.
[show abstract][hide abstract] ABSTRACT: Heat shock protein 27 (hsp27) is expressed by squamous cell carcinoma of the uterine cervix. Results from an earlier study by our group indicted that hsp27 may be a diagnostic marker for cervical intraepithelial neoplasia (CIN) and carcinoma. p16 expression is known to be elevated in intraepithelial uterine cervical cancer and grades 2 and 3 lesions (CIN2, CIN3), but has also been reported to be negative in 5-20% of cervical cancer and CIN lesions. The aim of our study was to confirm immunohistochemically the expression of hsp27 and p16 in cervical lesions. Formalin-fixed, paraffin-embedded cervical tissue specimens obtained between 2002 and 2010 were investigated for hsp27 and p16 expression. Positive staining was detected for hsp27 in 63% of normal cervical tissues, 47% of CIN1 lesions, 75% of CIN2 lesions, 92% of CIN3 lesions, and 100% of squamous cell carcinomas (SCC); the corresponding rates for p16 positivity were 29, 47, 67, 92, and 75%, respectively. Positive staining for both hsp27 and p16 was observed in 6% of normal cervical tissues and in 19% of CIN1, 18% of CIN2, 85% of CIN3, and 75% of SCC specimens. Hsp27 or p16 positivity had a sensitivity of 95.6 or 84.7% and a specificity of 37.2 or 70.5%, respectively, for the identification of CIN3 or SCC lesions; when both hsp27 and p16 were assessed, both the sensitivity and specificity were improved. In conclusion, both hsp27 and p16 immunohistochemistry is a useful tool for the diagnosis of CIN3 lesions or cervical SCC.
Human Cell 02/2012; 25(1):24-8. · 1.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: A case of a glomus tumor originating from the lung is reported. A 43-year-old female had undergone resection of a right lung tumor following a clinical diagnosis of carcinoid, sclerosing hemangioma, or other sarcoma. Histologically, the tumor comprised uniform small round to oval cells with centrally located nucleus, a clear cytoplasm, and apparent cell borders. The tumor also showed a focally hemangiopericytomatous pattern with irregularly branching or dilated vessels. Electron microscopy revealed smooth muscle differentiation of the tumor cells. Immunostaining further revealed that the tumor cells expressed smooth muscle actin, h-caldesmon, muscle specific actin (HHF-35), but not cytokeratin, epithelial membrane antigen, synaptophysin, or chromogranin A. Based on these findings, a diagnosis of primary pulmonary glomus tumor was established. Glomus tumors of the lung are very rare and only 21 cases have been reported to date. The histological features of the present tumor and the relevant literature are discussed.
[show abstract][hide abstract] ABSTRACT: 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.
Cancer Research 08/2010; 70(15):6384-92. · 8.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Various 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.
Core 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.
EC treatment induced nuclear foci of gammaH2AX, 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-, gammaH2AX-, 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.
High 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.
Breast cancer research: BCR 03/2010; 12(2):R17. · 5.87 Impact Factor
[show abstract][hide abstract] ABSTRACT: Gene-expression profiling classified breast cancer to intrinsic subtypes, including luminal A and B, HER2 positive, normal-breast-like, and basal-like tumors. Of these, basal-like tumors that express basal cytokeratins and that are negative for estrogen receptor alpha, progesterone receptor, and HER2 show the most aggressive phenotype with a poor prognosis. Analyses of clinical samples and basic research indicate that basal-like breast cancer is caused by deficiencies in the breast cancer susceptibility protein, BRCA1. Indeed, conditionally deleting BRCA1 from the mammary gland causes mice to develop basal-like cancers at high rates. One of the major functions of BRCA1 is DNA double-strand break (DSB) repair, and its failure to perform causes increased sensitivity of cells to DNA damage-inducing agents, such as PARP inhibitors, DNA cross-linkers, or topoisomerase inhibitors. Therefore, BRCA1 dysfunction could be a principal target for therapeutic application of basal-like breast cancer. Recently, significant progress has been made in understanding the BRCA1 cascade in response to DSBs, where ubiquitin polymer formation plays critical roles. Ubiquitination was indeed found to be an apparent early response of breast cancer to neoadjuvant treatment with epirubicin and cyclophosphamide. Deducing the role of BRCA1 ubiquitin E3 ligase activity in this pathway is a critical challenge to further clarify its functional mechanism. In individualized treatment of breast cancer, evaluation of the DNA repair capacity by the BRCA1 pathway may be an important issue when determining proper treatment of basal-like breast cancer.
Breast Cancer 06/2009; 16(4):268-74. · 1.33 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Human pathology 09/2008; 40(1):41-9. · 3.03 Impact Factor
[show abstract][hide abstract] ABSTRACT: Neuroblastoma frequently shows spontaneous regression in which two distinct types of programmed cell death, ie, caspase-dependent apoptosis and H-Ras-mediated autophagic degeneration, have been suggested to play a key role. The current study was conducted to determine which of these cell suicide pathways predominated in this tumor regression. Periodic acid-Schiff (PAS) staining and immunostaining for H-Ras and for the full-length and cleaved forms of caspase-3, poly (ADP-ribose) polymerase (PARP), and lamin A were carried out on 55 archival tumor specimens. The incidence of caspase-dependent apoptosis in each tumor was quantified by cleaved lamin A staining and compared with clinicopathologic prognostic factors. Although a recent report has shown that neuroblastic cells undergoing autophagic degeneration were readily detectable by PAS and H-Ras staining, we could not confirm this result in any of our samples with the exception of one tumor. Instead, many of our neuroblastoma samples showed nonspecific PAS and Ras staining in areas of necrosis, suggesting that autophagic "degeneration" indeed corresponds to coagulation necrosis or oncosis. Unexpectedly, the incidence of caspase-dependent apoptosis was significantly correlated with indicators of a poor prognosis in these tumors, including Shimada's unfavorable histology, MYCN amplification, and a higher mitosis-karyorrhexis index, but not with factors related to tumor regression such as clinical stage and mass screening. These results indicate that neither caspase-dependent apoptosis nor autophagic "degeneration" may be involved in spontaneous neuroblastoma regression. This suggests that other mechanisms, perhaps such as tumor maturation, may be responsible for this phenomenon.
American Journal of Surgical Pathology 03/2006; 30(2):249-57. · 4.87 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Febs Letters - FEBS LETT. 01/2001; 494(3):181-185.