[Show abstract][Hide abstract] ABSTRACT: Notch activity regulates tumor biology in a context-dependent and complex manner. Notch may act as an oncogene or a tumor-suppressor gene even within the same tumor type. Recently, Notch signaling has been implicated in cellular senescence. Yet, it remains unclear as to how cellular senescence checkpoint functions may interact with Notch-mediated oncogenic and tumor-suppressor activities. Herein, we used genetically engineered human esophageal keratinocytes and esophageal squamous cell carcinoma cells to delineate the functional consequences of Notch activation and inhibition along with pharmacological intervention and RNA interference experiments. When expressed in a tetracycline-inducible manner, the ectopically expressed activated form of Notch1 (ICN1) displayed oncogene-like characteristics inducing cellular senescence corroborated by the induction of G0/G1 cell-cycle arrest, Rb dephosphorylation, flat and enlarged cell morphology and senescence-associated β-galactosidase activity. Notch-induced senescence involves canonical CSL/RBPJ-dependent transcriptional activity and the p16(INK4A)-Rb pathway. Loss of p16(INK4A) or the presence of human papilloma virus (HPV) E6/E7 oncogene products not only prevented ICN1 from inducing senescence but permitted ICN1 to facilitate anchorage-independent colony formation and xenograft tumor growth with increased cell proliferation and reduced squamous-cell differentiation. Moreover, Notch1 appears to mediate replicative senescence as well as transforming growth factor-β-induced cellular senescence in non-transformed cells and that HPV E6/E7 targets Notch1 for inactivation to prevent senescence, revealing a tumor-suppressor attribute of endogenous Notch1. In aggregate, cellular senescence checkpoint functions may influence dichotomous Notch activities in the neoplastic context.Oncogene advance online publication, 16 June 2014; doi:10.1038/onc.2014.169.
[Show abstract][Hide abstract] ABSTRACT: Insulin-like growth factor binding protein 3 (IGFBP3), a hypoxia-inducible gene, regulates a variety of cellular processes including cell proliferation, senescence, apoptosis and epithelial-mesenchymal transition (EMT). IGFBP3 has been linked to the pathogenesis of cancers. Most previous studies focus upon proapoptotic tumor suppressor activities of IGFBP3. Nevertheless, IGFBP3 is overexpressed in certain cancers including esophageal squamous cell carcinoma (ESCC), one of the most aggressive forms of squamous cell carcinomas (SCCs). The tumor-promoting activities of IGFBP3 remain poorly understood in part due to a lack of understanding as to how the tumor microenvironment may influence IGFBP3 expression and how IGFBP3 may in turn influence heterogeneous intratumoral cell populations. Here, we show that IGFBP3 overexpression is associated with poor postsurgical prognosis in ESCC patients. In xenograft transplantation models with genetically engineered ESCC cells, IGFBP3 contributes to tumor progression with a concurrent induction of a subset of tumor cells showing high expression of CD44 (CD44H), a major cell surface receptor for hyaluronic acid, implicated in invasion, metastasis and drug resistance. Our gain-of-function and loss-of-function experiments reveal that IGFBP3 mediates the induction of intratumoral CD44H cells. IGFBP3 cooperates with hypoxia to mediate the induction of CD44H cells by suppressing reactive oxygen species (ROS) in an insulin-like growth factor-independent fashion. Thus, our study sheds light on the growth stimulatory functions of IGFPB3 in cancer, gaining a novel mechanistic insight into the functional interplay between the tumor microenvironment and IGFBP3.
American Journal of Cancer Research 01/2014; 4(1):29-41. · 4.17 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background:
Expression profiles of some microRNAs (miRNAs) were associated with clinicopathological findings in human prostate cancer (PC), but the relative expression of miRNAs among Gleason patterns (GPs) remains unclear. In this study, we investigated the expression of several known microRNAs in each GP of PC.
Formalin-fixed, paraffin embedded (FFPE) tissue samples were obtained from radical prostatectomy (RP) (patient set 1, n = 43, including (GP 3) n = 22, (GP 4) n = 35, and (GP 5) n = 12) and needle biopsy (patient set 2, n = 10, (GP 4) n = 10). Cancer tissues from each GP and adjacent normal counterparts were separately collected using laser-captured microdissection (LCM). Real-time RT-PCR was performed to determine the relative expression of miRNAs, including miR-31-5p, -34c-5p, -96-5p, -182-5p, -183-5p, -205-5p, -221-3p, and -222-3p, which were currently reported to be involved in PC progression.
In radical prostatectomy samples, relative expression of miR-31-5p, miR-34c-5p, and miR-205-5p in any GP was significantly decreased compared to normal counterpart. However, no significant difference was detected among GP 3, GP 4, and GP 5. Meanwhile, in the same GP4, expression of miR-31-5p miR-182-5p, and miR-205-5p in cancer tissues obtained from high grade cancer was significantly higher than those obtained from intermediate grade cancer. Validation study using biopsy samples revealed that the relative expression of miR-182-5p was statistically higher in high grade cancer even in same GP4.
We confirmed the expression of miR-182-5p depended on the cancer grade even in same GP 4. Expression of miRNA associated with Gleason grading system may contribute to more accurate preoperative cancer risk evaluation.
The Prostate 06/2013; 73(8). DOI:10.1002/pros.22626 · 3.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive forms of squamous cell carcinomas. Common genetic lesions in ESCC include p53 mutations and EGFR overexpression, both of which have been implicated in negative regulation of Notch signaling. In addition, cyclin D1 is overexpressed in ESCC and can be activated via EGFR, Notch and Wnt signaling. To elucidate how these genetic lesions may interact during the development and progression of ESCC, we tested a panel of genetically engineered human esophageal cells (keratinocytes) in organotypic 3D culture (OTC), a form of human tissue engineering. Notch signaling was suppressed in culture and mice by dominant negative Mastermind-like1 (DNMAML1), a genetic pan-Notch inhibitor. DNMAML1 mice were subjected to 4-Nitroquinoline 1-oxide-induced oral-esophageal carcinogenesis. Highly invasive characteristics of primary human ESCC were recapitulated in OTC as well as DNMAML1 mice. In OTC, cyclin D1 overexpression induced squamous hyperplasia. Concurrent EGFR overexpression and mutant p53 resulted in transformation and invasive growth. Interestingly, cell proliferation appeared to be regulated differentially between those committed to squamous-cell differentiation and those invading into the stroma. Invasive cells exhibited Notch-independent activation of cyclin D1 and Wnt signaling. Within the oral-esophageal squamous epithelia, Notch signaling regulated squamous-cell differentiation to maintain epithelial integrity, and thus may act as a tumor suppressor by preventing the development of a tumor-promoting inflammatory microenvironment.
American Journal of Cancer Research 08/2012; 2(4):459-75. · 4.17 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Insulin-like growth factor binding protein (IGFBP)-3 regulates cell proliferation and apoptosis in esophageal squamous cell carcinoma (ESCC) cells. We have investigated how the hypoxic tumor microenvironment in ESCC fosters the induction of IGFBP3. RNA interference experiments revealed that hypoxia-inducible factor (HIF)-1α, but not HIF-2α, regulates IGFBP3 mRNA induction. By chromatin immunoprecipitation and transfection assays, HIF-1α was found to transactivate IGFBP3 through a novel hypoxia responsive element (HRE) located at 57 kb upstream from the transcription start site. Metabolic labeling experiments demonstrated hypoxia-mediated inhibition of global protein synthesis. 7-Methyl GTP-cap binding assays suggested that hypoxia suppresses cap-dependent translation. Experiments using pharmacological inhibitors for mammalian target of rapamycin (mTOR) suggested that a relatively weak mTOR activity may be sufficient for cap-dependent translation of IGFBP3 under hypoxic conditions. Bicistronic RNA reporter transfection assays did not validate the possibility of an internal ribosome entry site as a potential mechanism for cap-independent translation for IGFBP3 mRNA. Finally, IGFBP3 mRNA was found enriched to the polysomes. In aggregate, our study establishes IGFBP3 as a direct HIF-1α target gene and that polysome enrichment of IGFBP3 mRNA may permit continuous translation under hypoxic conditions.
The FASEB Journal 03/2012; 26(6):2620-30. DOI:10.1096/fj.11-198598 · 5.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hepatocyte growth factor (HGF) is a multifunctional molecule that acts as mitogen, motogen, and/or morphogen in a variety of cells. MET, a specific receptor tyrosine kinase for HGF, is upregulated in various tumors including squamous cell carcinoma of the human head and neck (HNSCC), but how HGF affects the expression of downstream functional genes has not yet been elucidated in detail. In the present study, we examined the expression of microRNA (miRNA), non-coding small RNA that regulate cell proliferation and functions by interfering with the translation of target mRNA, with or without HGF stimulation in HNSCC cell line HSC3. Among several miRNAs, in which the expression was altered after HGF stimulation, we focused on miR-200c and miR-27b, both of which were drastically downregulated after HGF stimulation. Expression of ZEB1, a target mRNA for miR-200c, was upregulated 3 and 6 h after HGF stimulation, and that of E-cadherin, a downstream molecule of ZEB1, was downregulated 12 h after HGF stimulation. Expression of ST14/matriptase, an enzyme for extracellular matrix (ECM) degradation and HGF activation and a target mRNA for miR-27b, was drastically upregulated in the protein level after HGF stimulation, although it was not statistically altered in the mRNA level. These results suggest that miR-200c and miR-27b downregulated by HGF might play an important role in epithelial-mesenchymal transition mediated by ZEB1/E-cadherin and ECM degradation and HGF autoactivation mediated by ST14/matriptase, respectively. Altered expression of miRNA directly regulated by HGF might contribute enhanced progressive and invasive characteristics of HNSCC by regulating the translation of HGF-induced functional molecules.
Cancer Science 09/2011; 102(12):2164-71. DOI:10.1111/j.1349-7006.2011.02096.x · 3.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Zinc finger E-box-binding (ZEB) proteins ZEB1 and ZEB2 are transcription factors essential in TGF-β-mediated senescence, epithelial-to-mesenchymal transition (EMT), and cancer stem cell functions. ZEBs are negatively regulated by members of the miR-200 microRNA family, but precisely how tumor cells expressing ZEBs emerge during invasive growth remains unknown. Here, we report that NOTCH3-mediated signaling prevents expansion of a unique subset of ZEB-expressing cells. ZEB expression was associated with the lack of cellular capability of undergoing NOTCH3-mediated squamous differentiation in human esophageal cells. Genetic inhibition of the Notch-mediated transcriptional activity by dominant-negative Mastermind-like 1 (DNMAML1) prevented squamous differentiation and induction of Notch target genes including NOTCH3. Moreover, DNMAML1-enriched EMT-competent cells exhibited robust upregulation of ZEBs, downregulation of the miR-200 family, and enhanced anchorage-independent growth and tumor formation in nude mice. RNA interference experiments suggested the involvement of ZEBs in anchorage-independent colony formation, invasion, and TGF-β-mediated EMT. Invasive growth and impaired squamous differentiation were recapitulated upon Notch inhibition by DNMAML1 in organotypic three-dimensional culture, a form of human tissue engineering. Together, our findings indicate that NOTCH3 is a key factor limiting the expansion of ZEB-expressing cells, providing novel mechanistic insights into the role of Notch signaling in the cell fate regulation and disease progression of esophageal squamous cancers.
Cancer Research 09/2011; 71(21):6836-47. DOI:10.1158/0008-5472.CAN-11-0846 · 9.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mucinous cystic neoplasm (MCN) of the pancreas is characterized by mucin-producing columnar epithelium and an ovarian-type stroma. It occurs almost exclusively in women and is almost always located in the pancreatic body or tail. Here, we report a case of large MCN located in the pancreatic head but not in the body nor tail in a 32-year-old pregnant woman, which was thought to have grown rapidly during pregnancy. It was ruptured at 34 weeks of gestation and the patient was admitted to the emergency department of the University of Fukui Hospital with an acute abdomen. Emergency cesarean section followed by pancreaticoduodenectomy was performed. The tumor consisted of many small cysts lined by a single-layer of mucinous epithelium with papillary growth and partial solid parts showing invasive growth and sarcomatoid changes, indicating mucinous cystic neoplasm with an associated invasive carcinoma (previously referred as mucinous cystadenocarcinoma). Thickened septa revealed ovarian-type stroma strongly positive for α-inhibin and partly positive for progesterone receptor immunohistochemically. We also review and discuss previous reports of MCNs including those with an associated invasive carcinoma in pregnant patients.
Pathology International 01/2011; 61(1):28-33. DOI:10.1111/j.1440-1827.2010.02609.x · 1.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: MicroRNAs (miRNAs) are non-coding small RNAs that regulate cell proliferation and functions by interfering with the translation of target mRNAs. Altered expression of miRNA is known to induce various human malignancies. We examined the expression of miRNAs in squamous cell carcinoma of human head and neck (HNSCC) and esophagus (ESCC), compared to that in normal squamous epithelia as well as malignancies of other organs. Microarray analysis showed up-regulation of miR-21, miR-16 and miR-30a-5p in HNSCC and ESCC cell lines compared to normal squamous epithelial cell lines, and consistent high expression of miR-205 and let-7a in both normal and malignant squamous epithelial cell lines. Validation study using real-time quantitative RT-PCR in formalin-fixed paraffin-embedded cancer tissues and paired normal epithelia obtained by Laser-captured microdissection revealed that miR-205 showed highest expression in both malignant and benign squamous epithelia, although it was less expressed in cell lines and tissues other than squamous epithelia. MiR-21, which is an oncogenic miRNA in various malignancies, was also up-regulated in HNSCC and ESCC compared to paired normal squamous epithelia. These results suggest that miR-205 might be a specific marker miRNA of both normal and malignant squamous epithelia, while miR-21 might be a putative oncogenic miRNA in HNSCC and ESCC.
[Show abstract][Hide abstract] ABSTRACT: We report 2 patients showing invasion of aspergillosis into the central nerve system (CNS). Patient 1, an 81-year-old woman, underwent surgery for sphenoidal sinusitis. She developed cerebral infarction with unconsciousness on 12th postoperative day. CSF examination demonstrated pleocytosis with increased protein and aspergillus antigen. She was diagnosed as having invasion of aspergillosis into the CNS, and was treated with voriconazole. Her clinical manifestations and CSF findings markedly improved. However, the effects of voriconazole gradually attenuated and she demonstrated recurrence of the cerebral infarction. After 2 months, she died of systemic aspergillosis and sepsis. Autopsy studies. Severe atherosclerotic changes with calcification were demonstrated in the bilateral carotid and basilar arteries, and many aspergillus were clustered in the vessel walls. Granulomatous inflammatory lesions with aspergillus were also demonstrated in the area surrounding the chiasm. There were no massive infarcts or bleeding in the brain, but multiple small infarcts were present. Patinet 2, a 64-year-old man, showing bilateral visual loss, was receiving treatment with corticosteroids under a diagnosis of optic neuritis. Two weeks later, he developed cerebral infarction. CSF examination showed pleocytosis with increased protein and aspergillus antigen. He was diagnosed as having invasive aspergillosis from the sphenoidal sinusitis into the CNS. He was treated with voriconazole, and unconsciousness and CSF findings improved transiently. However, he developed a recurrence of the brain infarction and pneumonia and finally died 6 months later. Treatment by voriconazole was definitely effective in both patients, but both patients died of recurrent cerebral infarction, possibly due to resistance for voriconazole, or developing multicellular filamentous biofilms. Voriconazole is recommended as the first choice of antifungal agents for aspergillosis. Aspergillus infection is strongly invasive into arterial vessels. It is important to consider the possible occurrence of cerebrovascular disease when treating invasion of aspergillosis into the CNS.