Sumitaka Yamanaka

Biopsy with Thermally‐Responsive Untethered Microtools

Thermally activated, untethered microgrippers can reach narrow conduits in the body and be used to excise tissue for diagnostic analyses. As depicted in the figure, the feasibility of an in vivo biopsy of the porcine bile duct using untethered microgrippers is demonstrated.

MicroRNA down-regulated in human cholangiocarcinoma control cell cycle through multiple targets involved in the G1/S checkpoint

MicroRNAs (miRs) recently emerged as prominent regulators of cancer processes. In the current study we aimed at elucidating regulatory pathways and mechanisms through which miR‐494, one of the miR species found to be down‐regulated in cholangiocarcinoma (CCA), participates in cancer homeostasis. miR‐494 was identified as down‐regulated in CCA based on miR arrays. Its expression was verified with quantitative real‐time reverse‐transcription polymerase chain reaction (qRT‐PCR). To enforce miR expression, we employed both transfection methods, as well as a retroviral construct to stably overexpress miR‐494. Up‐regulation of miR‐494 in cancer cells decreased growth, consistent with a functional role. mRNA arrays of cells treated with miR‐494, followed by pathway analysis, suggested that miR‐494 impacts cell cycle regulation. Cell cycle analyses demonstrated that miR‐494 induces a significant G1/S checkpoint reinforcement. Further analyses demonstrated that miR‐494 down‐regulates multiple molecules involved in this transition checkpoint. Luciferase reporter assays demonstrated a direct interaction between miR‐494 and the 3′‐untranslated region of cyclin‐dependent kinase 6 (CDK6). Last, xenograft experiments demonstrated that miR‐494 induces a significant cancer growth retardation in vivo. Conclusion: Our findings demonstrate that miR‐494 is down‐regulated in CCA and that its up‐regulation induces cancer cell growth retardation through multiple targets involved in the G1‐S transition. These findings support the paradigm that miRs are salient cellular signaling pathway modulators, and thus represent attractive therapeutic targets. miR‐494 emerges as an important regulator of CCA growth and its further study may lead to the development of novel therapeutics. (HEPATOLOGY 2011)

Coordinated effects of microRNA-494 induce G2/M arrest in human cholangiocarcinoma

MicroRNA (miRs) have emerged as salient regulators in cancer homeostasis and, recently, as putative therapeutics. Cholangiocarcinomas (CCA) are aggressive cancers with survival usually measured in months. mRNA arrays followed by pathway analysis revealed that miR-494 is a major modulator of the cell cycle progression from gap 2 (G₂) to mitosis (M). We performed fluorescence activated cell sorting (FACS) as well as differential interference contrast (DIC) microscopy, and confirmed that miR-494 induces a significant arrest in G₂/M in CCA cells. Furthermore, we verified that miR-494 modulates the protein level of six genes involved in the G₂/M transition: Polo-like Kinase 1 (PLK1), pituitary tumor-transforming gene 1 (PTTG1), Cyclin B1 (CCNB1), cell-division cycle 2 (CDC2), cell-division cycle 20 (CDC20) and topoisomerase II α (TOP2A). Next, we identified direct binding of miR-494 to the open reading frame (ORF) and downregulation of PTTG1 and TOP2A. In summary, our findings suggest that miR-494 has a global regulatory role in cell cycle progression, exerted by concerted effects on multiple proteins involved in gap 1 (G₁) to synthesis (S), as described previously, as well as G₂ to M progression. Therefore, it appears that the simultaneous effects of a single miR species on multiple targets along the same canonical pathway is advantageous for the usage of miRs as therapeutics. In addition, our data suggest that miRs act within a narrow range. miR expression above the upper threshold does not appear to induce further effects, which is reassuring in terms of off-target effects of miR surrounding noncancerous tissue.

MicroRNA-224 negatively regulates p21 expression during late neoplastic progression in inflammatory bowel disease

Background:The development of colon cancer represents a major complication in patients with inflammatory bowel disease (IBD). The importance of microRNAs (miRs) in carcinogenesis is becoming clearer because miRs have been implicated in the regulation of cancer-related cellular processes to include apoptosis, differentiation, cell cycle progression, and immune function. In the current study, we sought to identify miR dysregulation specific to progression along the normal-inflammation-cancer axis in colonic specimens from patients with IBD. Methods:MiR microarrays and quantitative reverse transcription PCR were used to detect and confirm dysregulated miRs. Receiver operating characteristic curve analysis was applied to evaluate the potential use of miR-224 as a neoplastic disease marker in IBD. For miR-224 target messenger RNA (mRNA) identification, mRNA microarrays were employed in combination with bioinformatic analyses, Western blotting, and luciferase activity measurements. Results:We identified 30 miRs that were differentially expressed between chronically inflamed mucosae and cancers arising from IBD tissues. MiR-224 levels increased successively at each stage of IBD progression and accurately discriminated cancers from normal or chronically inflamed IBD tissues. Moreover, mRNA arrays combined with bioinformatic analyses suggested the participation of miR-224 in cell cycle regulation. Subsequently, cell cycle experiments indicated that miR-224 regulates the G1-S checkpoint. Finally, in silico prediction analyses, confirmed by Western blotting and luciferase assays, identified p21 as a specific direct mRNA target of miR-224. Conclusions:These findings reveal miR dysregulation specific to IBD-associated colorectal carcinoma. MiR-224 is overexpressed in IBD cancers and targets p21, a key cell cycle regulator. Moreover, these results establish the participation of miR-224 in IBD carcinogenesis.

Silencing of miR-370 in Human Cholangiocarcinoma by Allelic Loss and Interleukin-6 Induced Maternal to Paternal Epigenotype Switch

Cholangiocarcinoma (CCA) is a highly lethal malignant tumor arising from the biliary tract epithelium. Interleukin-6 (IL-6) is a major mediator of inflammation and contributor to carcinogenesis within the biliary tree. Previous studies suggested that enforced IL-6 contributes to cholangiocarcinogenesis through hypermethylation of several genes implicated in CCA. However, the precise mechanisms of IL-6 effects in CCA remain unclear. We now demonstrate that microRNA (miR)-370 is underexpressed in a large cohort of human CCA vs. normal liver tissues. In addition, we show that IL-6 induces a time-dependent silencing of miR-370. In addition, demethylation of CCA cells results in upregulation of miR-370. Furthermore, we demonstrate that miR-370 is imprinted, and that the Intergenic Differentially Methylated Region (IG-DMR) responsible for imprinting regulation of this genomic locus is hypermethylated in response to IL-6 treatment. In addition, the IG-DMR is hypermethylated in human CCA specimens compared to normal matched controls, in the same location as the IL-6 induced hypermethylation. Finally, miR-370 was found to regulate WNT10B in luciferase as well as western blotting experiments. Our data indicate that the paternal allele of miR-370 is normally silenced through genomic imprinting and that the overexpression of IL-6 in CCA effectively suppresses the expression of miR-370 from the maternal allele, lending support to the theory that miR-370 silencing in human CCA follows a classic two-hit mechanism.

A Total Pleural Covering for Lymphangioleiomyomatosis Prevents Pneumothorax Recurrence

Background Spontaneous pneumothorax is a major and frequently recurrent complication of lymphangioleiomyomatosis (LAM). Despite the customary use of pleurodesis to manage pnenumothorax, the recurrence rate remains high, and accompanying pleural adhesions cause serious bleeding during subsequent lung transplantation. Therefore, we have developed a technique of total pleural covering (TPC) for LAM to wrap the entire visceral pleura with sheets of oxidized regenerated cellulose (ORC) mesh, thereby reinforcing the affected visceral pleura and preventing recurrence. Methods Since January 2003, TPC has been applied during video-assisted thoracoscopic surgery for the treatment of LAM. The medical records of LAM patients who had TPC since that time and until August 2014 are reviewed. Results TPC was performed in 43 LAM patients (54 hemithoraces), 11 of whom required TPC bilaterally. Pneumothorax recurred in 14 hemithoraces (25.9%) from 11 patients (25.6%) after TPC. Kaplan-Meier estimates of recurrence-free hemithorax were 80.8% at 2.5 years, 71.7% at 5 years, 71.7% at 7.5 years, and 61.4% at 9 years. The recurrence-free probability was significantly better when 10 or more sheets of ORC mesh were utilized for TPC (P = 0.0018). TPC significantly reduced the frequency of pneumothorax: 0.544 ± 0.606 episode/month (mean ± SD) before TPC vs. 0.008 ± 0.019 after TPC (P<0.0001). Grade IIIa postoperative complications were found in 13 TPC surgeries (24.1%). Conclusions TPC successfully prevented the recurrence of pneumothorax in LAM, was minimally invasive and rarely caused restrictive ventilatory impairment.

Biologic tissue sampling with untethered microgrippers.

Even with advances in non-invasive diagnosis of diseases such as cancer or inflammatory diseases, tissue biopsy coupled with histopathologic examination remains the gold standard in establishing an accurate diagnosis.1–2 Generally, effective tissue diagnosis is fundamentally based on biopsying lesions that are suspicious based on visual inspection. Nevertheless, numerous mucosal conditions, such as dysplasia in ulcerative colitis or Barrett’s esophagus, are not always readily recognizable visually, and thus mandate surveillance protocols that involve random biopsies.3 To effectively sample large organs such as the colon, numerous biopsies are required,4 which can be time consuming. Moreover, due to the size of current forceps and the associated mucosal trauma and to the serial operation of the forceps, the number of random biopsies that can be realistically carried out is limited. Consequently, the current standard of care for cancer surveillance in ulcerative colitis patients is performed with at least 33 sequential biopsies (4-quadrant biopsies every 10 cm), which we estimate cumulatively samples less than 0.3% of colonic mucosa. The low sampling coverage may be ineffective at detecting precancerous or cancerous lesions, especially for early, small lesions that are also the most treatable.

Left-Sided Catamenial Pneumothorax with Thoracic Endometriosis and Bullae in the Alveolar Wall

Catamenial pneumothorax (CP) is generally caused by intraperitoneal air leaking from the uterus into the thoracic cavity via a defect in the endometrial tissue of the diaphragm and is usually detected in the right thorax. We report a case of left-sided CP caused by endometriosis in the visceral pleura and with no abnormal findings in the diaphragm. A 33-year-old female patient presented at the end of a course of low-dose contraceptive pills for pelvic endometriosis, with spontaneous pneumothorax in the left chest. Chest CT revealed a bulla in the left upper lung lobe. The patient underwent partial resection of the lung. Immunohistochemistry confirmed the presence of endometrial stromal tissue in the visceral pleura and confirmed this as the cause of pneumothorax since there were no observable abnormalities in the diaphragm. This case suggests that immunohistochemical examination of patients with spontaneous pneumothorax can detect alternative endometrial lesions.

Two Kinds of Cystic Lung Lesions with Pulmonary Lymphangioleiomyomatosis in a Male.

A 34-year-old male with frequent recurrence of right pneumothorax was admitted to our hospital. He was a current smoker and outwardly male without genital aplasia. He was diagnosed as tuberous sclerosis complex (TSC) at 2 year-old and underwent transcatheter arterial embolization for right renal hemorrhage due to renal tumor 2 years ago. Chest Computed Tomography showed that he had multiple tiny round cystic lesions with thin wall in both lungs. The recurrent pneumothorax was expected to be associated with TSC-Lymphangioleiomyomatosis (LAM). Video-assisted thoracic surgery was successfully performed. The operative and histological findings revealed that the bullae were classified into two groups; emphysematous bullae and bullae due to LAM. His postoperative course was uneventful. TSC-LAM is extremely rare, but in some cases the clinical recognition might be escaped due to subtle findings of bullae in early LAM, resulting in diagnosis as spontaneous pneumothorax.

Abstract 5284: A microRNA downregulated in human cholangiocarcinoma induces G2/M arrest through multiple targets.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: Cholangiocarcinomas (CCA) are cancers with dismal prognosis, thought to arise from epithelial cells lining the biliary tree. An improved understanding of the pathogenesis of CCA, as well as novel diagnostic and therapeutic approaches are direly needed. MicroRNAs (miRs) are short, single-stranded sequences of RNA, that were recently demonstrated to play a major role in the regulation of virtually all cellular processes. Several previous studies identified miRs that are dysregulated in CCA as well as distinct roles played by miRs in CCA genesis or progression. Methods: We performed microRNA-arrays on 5 normal biliary duct epithelias and 5 CCAs that had been obtained at surgery. After data analysis, we selected mir-494 for all subsequent analysis among top 5 miRs. To confirm the initial miR array data, quantitative real time RT-PCR analysis was performed using 12 human CCA as well as 5 normal cholangiocyte specimens. And then, we transfected 2 CCA cell lines with miR-494 and a non-specific mimic, respectively, and performed mRNA arrays to identify in an unbiased fashion the genes whose expression is downregulated by miR-494. Furthermore, we performed fluorescence activated cell sorting, as well as differential interference contrast microscopy. Results: miR-494 is significantly downregulated in human CCA specimens. Cells transfected with miR-494 showed a significant decrease in growth in the cell proliferation assay. As for the results of mRNA array, Ingenuity Pathway Analysis also indicated that miR-494 appears to coordinately affect several genes involved in the Mitotic Roles of Polo-like Kinase and Cell cycle: G2/M Checkpoint Regulation canonical pathways. We verified that miR-494 modulates the protein level of six genes involved in the G2/M transition. Next, we identified direct binding of miR-494 to the open reading frame (ORF), and downregulation of PTTG1 and TOP2A. Conclusions: our findings suggest that miR-494 has a global regulatory role in cell cycle progression. Therefore, it appears that the simultaneous effects of a single miR species on multiple targets along the same canonical pathway is advantageous for the usage of miRs as therapeutics. Citation Format: Sumitaka Yamanaka, Nathaniel R. Campbell, Scot C. Kuo, Esteban Mezey, Anirban Maitra, Florin Selaru. A microRNA downregulated in human cholangiocarcinoma induces G2/M arrest through multiple targets. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5284. doi:10.1158/1538-7445.AM2013-5284