Makoto Sano

Myogenesis in Wilms Tumors Is Associated with Mutations of the WT1 Gene and Activation of Bcl-2 and the Wnt Signaling Pathway

Wilms tumors with WT1 mutations [WT1(−)] have a stromal-predominant histology with varying extents of rhabdomyogenesis. These tumors also frequently have mutations in the beta-catenin gene (CTNNB1). We have investigated the molecular events that may explain the origins of rhabdomyogenesis in WT1(−) tumors. Of 35 Wilms tumors, we identified 12 with WT1 mutations, of which 9 carried CTNNB1 mutations. We compared WT1 wild-type tumors [WT1(+)] with WT1(−) tumors for histological features, localization of beta-catenin, Bcl-2 expression, and apoptosis using an in-situ end-labeling technique. WT1(+) tumors showed triphasic and blastemal- and epithelial predominant-histology. Expression of WT1, beta-catenin, and Bcl-2 recapitulated those of normal kidney epithelial development. Localization of beta-catenin was observed in the cytoplasm and cytoplasmic membrane of early glomerular epithelial structures. Bcl-2 is also expressed in condensing blastema and early glomerular epithelial structures which had little apoptosis. WT1(−) tumors, regardless of whether CTNNB1 mutations were detected or not, showed a stromal-rich phenotype with abundant expression of beta-catenin in the nucleus of the rhabdomyoblasts. Bcl-2 was expressed in rhabdomyoblasts, but not in blastemal cells undergoing apoptosis, suggesting that WT1 regulates Bcl-2 positively in the epithelial pathway, but negatively in the myogenic pathway. These data indicate that mutations in WT1 might alter the Wnt signaling pathway and Bcl-2 related-apoptosis. In WT1(−) tumors, the nuclear accumulation of beta-catenin and Bcl-2 expression are associated with rhabdomyogenesis, and dysregulation of Bcl-2 may be a mechanism by which the histogenesis (loss of blastemal component, muscle differentiation) may be explained.

Activated Wnt Signaling in Stroma Contributes to Development of Pancreatic Mucinous Cystic Neoplasms

BACKGROUND & AIMS Pancreatic mucinous cystic neoplasm (MCN), a cystic tumor of the pancreas that develops most frequently in women, is a potential precursor to pancreatic ductal adenocarcinoma. MCNs develop primarily in the body and tail of the pancreas and are characterized by the presence of a mucinous epithelium and ovarian-like subepithelial stroma. We investigated the involvement of Wnt signaling in KRAS-mediated pancreatic tumorigenesis and development of MCN in mice, and Wnt activation in human MCN samples. METHODS LSL-Kras(G12D), Ptf1a-cre mice were crossed with elastase-tva mice to allow for introduction of genes encoded by the replication-competent avian sarcoma-leukosis virus long-terminal repeat with splice acceptor viruses to pancreatic acinar cells and acinar cell progenitors, postnatally and sporadically. Repeat with splice acceptor viruses that expressed Wnt1 were delivered to the pancreatic epithelium of these mice; pancreatic lesions were analyzed by histopathology and immunohistochemical analyses. We analyzed levels of factors in Wnt signaling pathways in 19 MCN samples from patients. RESULTS Expression of Wnt1 in the pancreatic acinar cells and acinar cell progenitors of female mice led to development of unilocular or multilocular epithelial cysts in the pancreas body and tail, similar to MCN. The cystic lesions resembled the estrogen receptor- and progesterone receptor-positive ovarian-like stroma of MCN, but lacked the typical mucinous epithelium. Activated Wnt signaling, based on nuclear localization of β-catenin, was detected in the stroma but not cyst epithelium. Wnt signaling to β-catenin was found to be activated in MCN samples from patients, within the ovarian-like stroma, consistent with the findings in mice. CONCLUSIONS Based on studies of mice and pancreatic MCN samples from patients, the canonical Wnt signaling pathway becomes activated and promotes development of the ovarian-like stroma to contribute to formation of MCNs.

Identification of a Novel Cancer-Testis Antigen CRT2 Frequently Expressed in Various Cancers Using Representational Differential Analysis

Purpose: Cancer-testis antigens are promising targets for cancer immunotherapy. Identification of additional cancer-testis antigens with frequent expression in various cancers was attempted using representational differential analysis (RDA) and immunogenicity evaluation. Experimental Design: cDNAs preferentially expressed in testis were enriched using RDA by subtraction between testis and normal tissues. Thirty clones showing cancer-testis–like expression based on EST database analysis were evaluated by reverse transcription-PCR. A potential antigen, CRT2, was identified and its expression was analyzed with a newly generated anti-CRT2 antibody. The immunogenicity of CRT2 was examined based on reactivity with serum immunoglobulin G (IgG) from cancer patients, using Western blot and ELISA analysis, and on in vitro induction of tumor-reactive CTLs from HLA-A24 transgenic mice and human peripheral blood lymphocytes. Results: CRT2 was expressed in elongated spermatids of testis among normal tissues and in various cancer cell lines and tissues. The recombinant CRT2 protein was recognized by serum IgG from patients with various cancers in Western blot and ELISA analyses. A CRT2-derived peptide was identified as an HLA-A24–restricted T-cell epitope that induced tumor-reactive CTLs. Conclusion: CRT2 was identified as a new cancer-testis antigen expressed in elongated spermatids of testis and in cancer tissues (particularly melanoma) that is recognized by serum IgG from cancer patients. An HLA-A24–restricted T-cell epitope capable of inducing tumor-reactive CTLs was identified, suggesting that CRT2 may be useful for cancer diagnosis and immunotherapy.

Overexpression of anti-apoptotic Mcl-1 in testicular germ cell tumours

Aims:  To determine the expression of Mcl‐1 in testicular germ cell tumours in order to clarify the role of this anti‐apoptotic factor in these tumours. Various members of the Bcl‐2 family have been implicated in the apoptotic mechanisms regulating germ cell apoptosis. Mcl‐1 is an anti‐apoptotic Bcl‐2 family member and has recently been reported to be related to the progression of malignancy; however, the involvement of Mcl‐1 in the development of germ cell tumours is still unknown.

Activation of WNT/β-Catenin Signaling Enhances Pancreatic Cancer Development and the Malignant Potential Via Up-regulation of Cyr61

Pancreatic ductal adenocarcinoma (PDAC), a poor prognostic cancer, commonly develops following activating mutations in the KRAS oncogene. Activation of WNT signaling is also commonly observed in PDAC. To ascertain the impact of postnatal activation of WNT-stimulated signaling pathways in PDAC development, we combined the Elastase-tva-based RCAS-TVA pancreatic cancer model with the established LSL-KrasG12D, Ptf1a-cre model. Delivery of RCAS viruses encoding β-cateninS37A and WNT1 stimulated the progression of premalignant pancreatic intraepithelial neoplasias (PanIN) and PDAC development. Moreover, mice injected with RCAS-β-cateninS37A or RCAS-Wnt1 had reduced survival relative to RCAS-GFP-injected controls (P < .05). Ectopic expression of active β-catenin, or its DNA-binding partner TCF4, enhanced transformation associated phenotypes in PDAC cells. In contrast, these phenotypes were significantly impaired by the introduction of ICAT, an inhibitor of the β-catenin/TCF4 interaction. By gene expression profiling, we identified Cyr61 as a target molecule of the WNT/β-catenin signaling pathway in pancreatic cancer cells. Nuclear β-catenin and CYR61 expression were predominantly detected in moderately to poorly differentiated murine and human PDAC. Indeed, nuclear β-catenin- and CYR61-positive PDAC patients demonstrated poor prognosis (P < .01). Knockdown of CYR61 in a β-catenin-activated pancreatic cancer cell line reduced soft agar, migration and invasion activity. Together, these data suggest that the WNT/β-catenin signaling pathway enhances pancreatic cancer development and malignancy in part via up-regulation of CYR61.

mTORC2 signaling drives the development and progression of pancreatic cancer

mTOR signaling controls several critical cellular functions and is deregulated in many cancers, including pancreatic cancer. To date, most efforts have focused on inhibiting the mTORC1 complex. However, clinical trials of mTORC1 inhibitors in pancreatic cancer have failed, raising questions about this therapeutic approach. We employed a genetic approach to delete the obligate mTORC2 subunit Rictor and identified the critical times during which tumorigenesis requires mTORC2 signaling. Rictor deletion resulted in profoundly delayed tumorigenesis. Whereas previous studies showed most pancreatic tumors were insensitive to rapamycin, treatment with a dual mTORC1/2 inhibitor strongly suppressed tumorigenesis. In late-stage tumor-bearing mice, combined mTORC1/2 and PI3K inhibition significantly increased survival. Thus, targeting mTOR may be a potential therapeutic strategy in pancreatic cancer. Cancer Res; 76(23); 6911-23. ©2016 AACR.

MicroRNAs of the mir-17~92 cluster regulate multiple aspects of pancreatic tumor development and progression

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy characterized by resistance to currently employed chemotherapeutic approaches. Members of the mir-17~92 cluster of microRNAs (miRNAs) are upregulated in PDAC, but the precise roles of these miRNAs in PDAC are unknown. Using genetically engineered mouse models, we show that loss of mir-17~92 reduces ERK pathway activation downstream of mutant KRAS and promotes the regression of KRASG12D-driven precursor pancreatic intraepithelial neoplasias (PanINs) and their replacement by normal exocrine tissue. In a PDAC model driven by concomitant KRASG12D expression and Trp53 heterozygosity, mir-17~92 deficiency extended the survival of mice that lacked distant metastasis. Moreover, mir-17~92-deficient PDAC cell lines display reduced invasion activity in transwell assays, form fewer invadopodia rosettes than mir-17~92-competent cell lines and are less able to degrade extracellular matrix. Specific inhibition of miR-19 family miRNAs with antagomirs recapitulates these phenotypes, suggesting that miR-19 family miRNAs are important mediators of PDAC cell invasion. Together these data demonstrate an oncogenic role for mir-17~92 at multiple stages of pancreatic tumorigenesis and progression; specifically, they link this miRNA cluster to ERK pathway activation and precursor lesion maintenance in vivo and identify a novel role for miR-19 family miRNAs in promoting cancer cell invasion.

Differential diagnosis of trichosporonosis using conventional histopathological stains and electron microscopy.

Obana Y, Sano M, Jike T, Homma T & Nemoto N
(2010) Histopathology56, 372–383

Mcl-1, an anti-apoptotic Bcl-2 family member, essentially overlaps with insulin-producing cells in neonatal nesidioblastosis

Dear Sirs, We report here that anti-apoptotic protein Mcl-1 is closely related to the abnormal development of insulin-producing cells in neonatal nesidioblastosis. Nesidioblastosis, first reported by Dr. Laidlaw in 1938, was identified as the proliferation of endocrine cells differentiating from the pancreatic ductal epithelium to form islets, but the term “nesidioblastosis” has recently been used for histopathological abnormalities with hyperinsulinemic and hypoglycemic events [4]. Mcl-1, one of the anti-apoptotic Bcl-2 family members, is dispersed in fetal and adult pancreatic islets [1, 2, 6], and Mcl-1-positive cells have a distribution closely correspondent to that of glucagon-producing cells [2]. We have recently shown that Mcl-1 transgenic mice develop islet cell hyperplasia or insulinoma [3], indicating that constitutional persistent expression of Mcl-1 causes abnormal proliferation or new development of insulin-inducing cells in various islet cell disorders. This suggests that Mcl-1 is involved in pathogenesis of nesidioblastosis. We have observed a neonate patient with intractable hyperinsulinemic and hypoglycemia whose histopathological findings showed typical features of diffuse nesidioblastosis. To clarify the anti-apoptotic role of Mcl-1 in nesidioblastosis, we carried out an immunohistochemical investigation using antibodies against Mcl-1 [5, 6] (1:50, rabbit polyclonal), insulin (1:400; rabbit polyclonal; DAKO Cytomation, Kyoto, Japan), glucagon (1:50; guinea pig polyclonal; DAKO Cytomation), and Ki-67 (1:50; mouse monoclonal, MIB-1; DAKO Cytomation). Staining was developed with a 3, 3′-diaminobenzidine (DAB) or True Blue (KPL, Gaithersburg, MD). Slides were counterstained with hematoxylin or nuclear fast red. As opposed to reported in the normal fetal and adult pancreas, the distribution of Mcl-1-positive cells almost matched that of insulin-positive cells (Fig. 1a, Mcl-1; b, insulin) and rarely overlapped with that of glucagon-positive cells in nesidioblastosis (Fig. 1c). Mcl-1 was also expressed at low levels in the pancreatic ducts and intercalated ducts, which highlighted ductulo-insular proliferative lesions (Fig. 1d). We next addressed the question of whether or not these Mcl-1-positive cells possess proliferative properties. We performed double immunostaining for Mcl-1 and Ki-67. Most of the proliferating Mcl-1-positive cells in the pancreatic islets were devoid of Ki-67 labeling (Fig. 1e). Lack of Ki-67 staining was also observed in the Mcl-1positive cells in the ductulo-insular proliferation (Fig. 1f). Thus, our results suggested that Mcl-1 expression contributes to development of new insulin-producing cells rather than cell proliferation. We have shown overexpression of Mcl-1 in the ectopic and proliferative insulin-producing cells of human nesidioblastosis, which is consistent with the Mcl-1 transgenic mice model [3]. As endocrine cells derived from progenitor Virchows Arch (2008) 452:469–470 DOI 10.1007/s00428-007-0567-4

Osteoclastogenesis in human breast carcinoma.

Dear Sir, We herein report the involvement of vascular endothelial growth factor (VEGF) expression and osteoclast-like giant cell (OGC) formation in human breast carcinoma. We believe that OGCs are osteoclasts derived from peripheral blood macrophages, while overexpression of VEGF in the carcinoma cells may play a pivotal role in the osteoclastogenesis. A 46-year-old Japanese woman had a 30-mm palpable mass at the upper outer quadrant of her left breast. She underwent ultrasonography and mammography. Mammography showed a high-density mass in her left upper outer quadrant, but neither spiculation nor microcalcification were observed. Moreover, an oval-shaped hypoechoic mass with a well-demarcated margin was detected by ultrasonography. These imaging findings suggested that the tumor mass was a benign lesion, and the patient was suspected of having a fibroadenoma. She underwent fine-needle aspiration from the breast lesion, from which the cytological diagnosis was a suspected carcinoma with OGCs. The patient underwent conservative surgery of the left breast with dissection of the tumor and lymph nodes. Gross macroscopic analysis of the resected surface of the 25 25 20-mm nodule showed a yellowish solid appearance with a well-circumscribed margin, but a part of the peripheral margin of the tumor appeared dark brownish. Microscopically, the major histological pattern of the tumor was invasive ductal carcinoma, papillotubular carcinoma, with OGCs being frequently observed in the fibroblastic or hemorrhagic vascular stroma as well as in abutted carcinoma nests or the glandular lumen (Fig. 1a). Moreover, severe hemorrhage and infiltration of hemosiderin-laden macrophages were observed in the dark-brownish and yellow-brownish lesions. Lymph-node metastasis was microscopically seen in one-fourteenth of excised lymph nodes, while carcinoma metastasis was accompanied by an OGC component. To determine the characteristics of the carcinoma and OGCs, we performed immunohistochemical analysis (Table 1). OGCs were immunoreactive for anti-CD68 (monoclonal, 1:80; Dako Japan) (Fig. 1b), but not for antibodies to epithelial markers, such as cytokeratin (monoclonal, 1:50; Dako Japan) and epithelial membrane antigen (EMA, monoclonal, 1:50; Dako Japan). These results suggest that OGCs are derived from macrophages. Progesterone receptor (monoclonal, 1:50; Dako Japan) was detected in the nuclei of carcinoma cells, whereas estrogen receptor (monoclonal, 1:50; Dako Japan) p53 (monoclonal, 1:100; Dako Japan) and c-erbB2 (monoclonal, 1:100; Nichirei Co.) were under the detectable level. Of the carcinoma nuclei, 22% were positive for a proliferation marker Ki-67 (monoclonal, 1:25; Dako Japan). These bodies of evidence indicated that the carcinoma cells were at a biologically low grade of malignancy compared with other subtypes of breast carcinomas. To elucidate the hypervascular stroma and OGC formation in breast carcinoma, we determined the expression of VEGF and VEGF receptors. An antibody to endoglin (CD105, monoclonal, 1:100; Dako, catalyzed signal amplification system) revealed dense angiogenesis in the stroma (Fig. 1c). Prominent VEGF (polyclonal, 1:100; Santa Cruz Biotechnology) was detected in the carcinoma cells and OGCs, but mononuclear macrophages were negative (Fig. 1d). Flt-1 (VEGFR-1, polyclonal, 1:100; Santa Cruz Biotechnology) was detected in the carcinoma cells and the round OGCs located at the carcinoma lumen, but not detected in the elongated OGCs seen adjacent to the periphery of carcinoma nest (Fig. 1e). M. Sano ()) · K. Kikuchi · M. Kobayashi · Y. Nakanishi · N. Nemoto Department of Pathology, Nihon University School of Medicine, 30-1 Ohyaguchi-kamimachi, Itabashi-ku, 173-8610 Tokyo, Japan e-mail: msano@med.nihon-u.ac.jp