Nasopharyngeal carcinoma (NPC) is an Epstein‐Barr virus (EBV)‐associated epithelial malignancy. The high expression of BART‐miRNAs (miR‐BARTs) during latent EBV infection in NPC strongly supports ...their pathological importance in cancer progression. Recently, we found that several BART‐miRNAs work co‐operatively to modulate the DNA damage response (DDR) by reducing Ataxia‐telangiectasia‐mutated (ATM) activity. In this study, we further investigated the role of miR‐BARTs on DDR. The immunohistochemical study showed that the DNA repair gene, BRCA1, is consistently down‐regulated in primary NPCs. Using computer prediction programs and a series of reporter assays, we subsequently identified the negative regulatory role of BART2‐3p, BART12, BART17‐5p and BART19‐3p in BRCA1 expression. The ectopic expression of these four miR‐BARTs suppressed endogenous BRCA1 expression in EBV‐negative epithelial cell lines, whereas BRCA1 expression was enhanced by repressing endogenous miR‐BARTs activities in C666‐1 cells. More importantly, suppressing BRCA1 expression in nasopharyngeal epithelial cell lines using miR‐BART17‐5p and miR‐BART19‐3p mimics reduced the DNA repair capability and increased the cell sensitivity to the DNA‐damaging chemotherapeutic drugs, cisplatin and doxorubicin. Our findings suggest that miR‐BARTs play a novel role in DDR and may facilitate the development of effective NPC therapies.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
miR-375 is a tumor-suppressive microRNA (miRNA) in gastric cancer (GC). However, its molecular mechanism remains unclear. The aim of this study is to comprehensively investigate how miR-375 is ...involved in Hippo pathway by targeting multiple oncogenes. miR-375 expression in gastric cancer cell lines and primary GC was investigated by qRT-PCR. The regulation of YAP1, TEAD4, and CTGF expression by miR-375 was evaluated by qRT-PCR, western blot, and luciferase reporter assays, respectively. The functional roles of the related genes were examined by siRNA-mediated knockdown or ectopic expression assays. The clinical significance and expression correlation analysis of miR-375, YAP1, and CTGF were performed in primary GCs. TCGA cohort was also used to analyze the expression correlation of YAP1, TEAD4, CTGF, and miR-375 in primary GCs. miR-375 was down-regulated in GC due to promoter methylation and histone deacetylation. miR-375 downregulation was associated with unfavorable outcome and lymph node metastasis. Ectopic expression of miR-375 inhibited tumor growth in vitro and in vivo. Three components of Hippo pathway, YAP1, TEAD4 and CTGF, were revealed to be direct targets of miR-375. The expression of three genes showed a negative correlation with miR-375 expression and YAP1 re-expression partly abolished the tumor-suppressive effect of miR-375. Furthermore, CTGF was confirmed to be the key downstream of Hippo-YAP1 cascade and its knockdown phenocopied siYAP1 or miR-375 overexpression. YAP1 nuclear accumulation was positively correlated with CTGF cytoplasmic expression in primary GC tissues. Verteporfin exerted an anti-oncogenic effect in GC cell lines by quenching CTGF expression through YAP1 degradation. In short, miR-375 was involved in the Hippo pathway by targeting YAP1-TEAD4-CTGF axis and enriched our knowledge on the miRNA dysregulation in gastric tumorigenesis.
Patients with colorectal cancer (CRC) have a high incidence of regional and distant metastases. Although metastasis is the main cause of CRC-related death, its molecular mechanisms remain largely ...unknown.
Using array-CGH and expression microarray analyses, changes in DNA copy number and mRNA expression levels were investigated in human CRC samples. The mRNA expression level of RASAL2 was validated by qRT-PCR, and the protein expression was evaluated by western blot as well as immunohistochemistry in CRC cell lines and primary tumors. The functional role of RASAL2 in CRC was determined by MTT proliferation assay, monolayer and soft agar colony formation assays, cell cycle analysis, cell invasion and migration and in vivo study through siRNA/shRNA mediated knockdown and overexpression assays. Identification of RASAL2 involved in hippo pathway was achieved by expression microarray screening, double immunofluorescence staining and co-immunoprecipitation assays.
Integrated genomic analysis identified copy number gains and upregulation of RASAL2 in metastatic CRC. RASAL2 encodes a RAS-GTPase-activating protein (RAS-GAP) and showed increased expression in CRC cell lines and clinical specimens. Higher RASAL2 expression was significantly correlated with lymph node involvement and distant metastasis in CRC patients. Moreover, we found that RASAL2 serves as an independent prognostic marker of overall survival in CRC patients. In vitro and in vivo functional studies revealed that RASAL2 promoted tumor progression in both KRAS/NRAS mutant and wild-type CRC cells. Knockdown of RASAL2 promoted YAP1 phosphorylation, cytoplasm retention and ubiquitination, therefore activating the hippo pathway through the LATS2/YAP1 axis.
Our findings demonstrated the roles of RASAL2 in CRC tumorigenesis as well as metastasis, and RASAL2 exerts its oncogenic property through LATS2/YAP1 axis of hippo signaling pathway in CRC.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Colorectal cancer (CRC) is a heterogeneous disease with complex mechanisms of pathogenesis. Classification systems have been proposed based on molecular features of tumors in clinical practice. Thus, ...more molecular markers associated with development and progression of CRC might serve as useful tools for early diagnosis even for providing more accurate molecular classification. Frequent gain of chromosome 8q was detected in CRC by array‐CGH and overexpression of exosome component 4 (EXOSC4) in this region was revealed by expression microarray analysis. Through qRT‐PCR and immunohistochemistry (IHC) analysis, EXOSC4 showed increased expression in CRC cell lines and clinical specimens. Higher expression of EXOSC4 was more often detected in left side, and correlated with BRAF wild type, MSI‐low or MSS, CIMP‐low, and MLH1‐no‐silence CRC patients. Functionally, EXOSC4 overexpression increased early tumorigenic capacity by promoting cell proliferation and monolayer colony formation, enhancing cell invasion and migration study and accelerating xenograft formation in nude mice. While EXOSC4 knockdown exhibited anti‐oncogenic role such as inhibiting cell proliferation and invasion. EXOSC4 inhibition also resulted in G1 phase cell cycle arrest. For the downstream signaling analysis, EXOSC4 was found to be involved in multiple signaling pathways such as cell cycle, p53 pathway and Wnt pathway. In summary, our findings demonstrated the oncogenic role of EXOSC4 in development and progression of CRC. Deep understanding of EXOSC4 as a potential diagnostic molecular biomarker will provide clinical translational potential for intervention therapy.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Abstract
Nasopharyngeal carcinoma (NPC) is an aggressive epithelial malignancy that has remarkably high incidence and mortality rates in Hong Kong and south China. It is well-known to be associated ...with Epstein-Barr virus (EBV) infection. Previous work in our team revealed that EBV-encoded miRNAs derived from the BARTs (miR-BARTs) were abundantly expressed in primary NPCs. They function in controlling apoptotic and immune responses in the infected cells during NPC development.
Most recently, we attempted to use in silico method to predict cellular targets of miR-BARTs. Results suggested that there were multiple putative miR-BART binding sites on the 3’UTRs of an important DNA double-strand breaks (DSBs) repair gene, Ataxia-Telangiectasia-Mutant (ATM). The down-regulation of ATM mRNA and protein had also been demonstrated in our local primary NPC samples. Although the interaction of miR-BARTs on each suggested putative binding site had not been completely validated, our preliminary data indicated at least three EBV-encoded miRNAs (BART5-5p, BART9-3p and BART14-3p) were involved in repressing ATM expression in NPCs. They could work either alone or cooperatively to control ATM expression in transient assays. Notably, ectopic expression of these three miR-BARTs could successfully suppress γ irradiation-induced ATM activity in two EBV-negative cell lines, NP69 and HeLa.
It had been reported that ATM null cells were defective to repair the DSB lesions and sensitive to the Poly(ADP-ribose) polymerase (PARP) inhibitor treatment in the presence of DNA-damaging agents. In contrast, the DSBs are effectively repaired in normal cells to retain genetic integrity. We believed that EBV infection, via miR-BARTs to reduce ATM activity and disrupt Homologous Recombination (HR) repair function, made the NPC cells sensitize to ionizing radiation and DNA-damaging agent treatment. Hence, the knowledge generated from this project is not only enhance our understanding of EBV biology, but also opens an avenue for the development of effective NPC therapies.
Citation Format: RAYMOND Wai-Ming LUNG, Tom Pok-Man Hau, Wing-Po Chak, Joanna Hung-Man Tong, Ken Hung-On Yu, Sai-Wah Tsao, Kevin Yuk-Lap Yip, Ka-Fai To, Kwok-Wai Lo. The role of Epstein-Barr virus-encoded miRNAs in ATM regulating DNA damage response in nasopharyngeal carcinoma. abstract. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1118.
Background: The study aims at evaluation of the efficacy of elective neck dissection as a staging and therapeutic procedure for N0 neck of early carcinoma of the oral tongue by whole organ serial ...sectioning.
Methods: There were 50 stage I and II patients. The neck dissection specimens were whole organ sectioned in 3-mm intervals for the evaluation of nodal metastasis.
Results: There were 18 (36%) patients with subclinical nodal metastasis. The total number of metastatic nodes were 31 (1%) among all 2,826 nodes being examined. The metastatic foci had a median size of 3 mm and occupied a median of 6% of the cross sectional area of the involved nodes. The ipsilateral level II nodes were the commonest (26%) site of metastasis. Metastatic nodes were present in 34% patients who had negative preoperative radiological assessment and in 20% patients who had negative intraoperative frozen section sampling of neck nodes. Patients with subclinical nodal metastasis had a high incidence of regional recurrence (62%) and low survival (27%) when postoperative radiotherapy was not given after elective neck dissection.
Conclusions: Ipsilateral level I,II,III neck dissection is an adequate diagnostic procedure for staging of the N0 neck of early oral tongue carcinoma. Its diagnostic role cannot be replaced by the available pre-operative radiological screening and intra-operative frozen section sampling. However, elective selective neck dissection is an effective but not adequate therapeutic procedure, and post-operative adjuvant radiotherapy and chemotherapy have to be considered for all pathologically positive necks.
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IJS, IMTLJ, KILJ, KISLJ, NUK, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Background: The aims of the study are three-dimensional analysis of mode and distance of local spread of oral tongue carcinoma.
Methods: The glossectomy specimens were examined in the coronal plane ...in 3 mm thickness section.
Results: There were 50 glossectomy specimens. The maximum spread was 1.8 cm. Ninety-six percent of specimens had local spread within 1.2 cm. The distance of spread was not correlated with tumor size, including the diameter, depth, and volume. The incidence of local recurrence was 27% with positive histological margin. Perineural infiltration was the most important prognostic factor for local recurrence and survival.
Conclusions: A minimum of 1.5-cm surgical resection margin is recommended. A smaller margin is not recommended as it has significant risk of local recurrence. A maximum of 2-cm surgical resection margin is recommended; larger margins will increase the surgical morbidity without a significant advantage of local control.
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IJS, IMTLJ, KILJ, KISLJ, NUK, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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