Ml Lung

Functional characterization of FANCD2 in esophageal squamous cell carcinoma

Introduction: Esophageal squamous cell carcinoma (ESCC) has an especially high incidence in Northern China, where there is evidence for a significant familial association. We performed targeted next-generation sequencing (NGS) analysis on familial ESCC germline samples compared to non-cancer controls from the same high-risk region and compiled a list of candidate cancer predisposition genes. Interestingly, genes related to the Fanconi Anemia (FA) - BRCA pathway are enriched in the list. Among these FA-BRCA genes, Fanconi anemia complementation group D2 (FANCD2) was one of the top candidates, as it also had a high frequency of somatic mutations in ESCC tumor specimens. Therefore, we aim to characterize the role of FANCD2 in tumor development and explore its translational value. Methods: We knocked out the FANCD2 gene in ESCC cell lines using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technique to evaluate its potential oncogenic function in ESCC. Cell proliferation was measured by a MTT 2D clonogenic assay in vitro. Subcutaneous injection of the FANCD2 knockout ESCC cells into BALB/c-nude mice in vivo was performed to assess its functional impact on tumorigenesis. The single cell gel electrophoresis/comet assay was used to investigate the genome stability. Results: The FANCD2 knockout efficiency was confirmed by western blotting. Surprisingly, in vitro functional analyses showed that ESCC cells with FANCD2 knockout survive, with a greatly reduced growth rate and colony-forming ability. Consistent with the in vitro data, ESCC cells with FANCD2 knockout form significantly smaller subcutaneous tumors in nude mice. By applying the comet assay to examine the genome integrity, ESCC cells with FANCD2 knockout show significantly greater damage to the genome. Conclusion: These results suggest that FANCD2 plays an important role in supporting ESCC tumor growth. We attribute this to its core function in DNA repair ability and genome integrity maintenance. Acknowledgement: We acknowledge the grant support from the Hong Kong Research Grants Council Collaborative Research Fund (C7031.15G to M.L.L.). Citation Format: Lisa Chan Lei, Valen Zhuoyou Yu, Lvwen Ning, Josephine Mun-Yee Ko, Li Dong Wang, Maria Li Lung. Functional characterization of FANCD2 in esophageal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1357.

Abstract 4417: The role of EBV infection in aerobic glycolysis in nasopharyngeal carcinoma

Accumulating evidence indicates that oncogenic viral protein exerts a crucial role in activating aerobic glycolysis during tumorigenesis, but the underlying mechanisms are largely undefined. The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) is a trans-membrane protein with potent cell signaling properties and has tumorigenic transformation property. Activation of NF-κB is a major signaling pathway mediating many downstream transformation properties of LMP1. Here we report that activation of mTORC1 by LMP1 is a key modulator for activation of NF-κB signaling to mediate aerobic glycolysis. NF-κB activation is involved for LMP1-induced upregulation of glucose transptor-1 (Glut-1) transcription and growth of nasopharyngeal carcinoma (NPC) cells. Collectively, blocking the activity of mTORC1 signaling effectively suppressed LMP1-induced NF-κB activation and Glut-1 transcription. Interfering NF-κB signaling has no effect on mTORC1 activity but effectively altered Glut-1 transcription. Luciferase promoter assay of Glut-1 also confirmed that Glut-1 is a direct target gene of NF-κB signaling. Furthermore, we demonstrated that the LMP1 C-terminal activating region (CTAR) 2 is the key domain involved in mTORC1 activation, mainly through IKKβ-mediated phosphorylation of TSC2 at Ser939. Depletion of Glut-1 effectively led to suppression of aerobic glycolysis, inhibition of cell proliferation, colony formation, and attenuation of tumorigenic growth property of LMP1-expressing nasopharyngeal epithelial (NPE) cells. These findings suggest that targeting the signaling axis of mTORC1/NF-κB/Glut-1 represents a novel therapeutic target against NPC. Acknowledgement: 96 800x600 This project was supported by the General Research Fund (HKU 779810M, 17120814 and 17161116), CRF equipment grant (1061402980, Health and Medical Research Fund of Hong Kong (12110782), AoE grant (AoE/M-06/08) and TBRS grant (T12-401/13-R). We thank Prof. Dongyan Jin (Department of Biochemistry, The University of Hong Kong) for the kind gifts of IKK related plasmids and Prof. Zhenguo Wu (Division of Life Science, The Hong Kong University of Science and Technology) for the discussion and interpretation of the data. We also thank Mr. Tony Chan for his technical support. Note: This abstract was not presented at the meeting. Citation Format: George S. Tsao, Jun Zhang, Lin Xia, C Tsang, Weitao Lin, Y Yip, W Deng, K Lo, M Lung. The role of EBV infection in aerobic glycolysis in nasopharyngeal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4417. doi:10.1158/1538-7445.AM2017-4417

Abstract 632: Establishment and characterization of xenografts and cell lines trom nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is a common cancer among ethnic Cantonese living in Hong Kong and southern China. It is closely associated with Epstein-Barr virus (EBV) infection. Unfortunately, there are very few representative xenografts and cell lines established from NPC available for investigation. Most of the NPC xenografts established have been passaged in immune deficient animals for over 20 years and may not be representative of the original NPC in patients. For in vitro NPC cell lines, there is only one cell line which retains the EBV genomes. Other NPC cell lines have all lost their EBV episomes. Furthermore, many of these NPC cell lines are found to be contaminated with genetic components from HeLa cells (HPV16 genome) which raised issues on their origins and limited their uses as NPC cells. There is great urgency in establishment new NPC cell lines both in vivo and in vitro for various research investigations and for the study of pathogenic role of EBV infection in NPC. We have carried out continuous efforts since 2010 to attempt establishment of new NPC xenografts and cell lines from surgical and biopsies NPC tissues. NPC tissues from resected recurrent NPC and biopsies from primary NPC were explanted to subrenal capsular sites and maintained for four months to one year to observe for growth of new NPC xenografts. Attempts to establish new NPC celles were also carried out. At present, we have successfully established 3 NPC xenografts (Xeno 23, Xeno 32, Xeno 47) which could be passages at subcutaneously sites and one in vitro NPC cells (NPC43) which harbors EBV genomes. The growth properties and genetic alterations of these newly established NPC cell lines have been characterized. Novel genetic alterations and growth signaling pathways were observed in these newly established NPC cell lines and may represent driver mutations and signaling pathways essential for progression of NPC. Profiling of EBV gene expression of these newly established NPC cell lines revealed predominant latent EBV infection and expression of EBV-encoded mRNA from the BART transcripts which are characteristic of EBV infection in NPC. The establishment of new NPC cells will contribute to investigate the pathogenesis of NPC and its intimate relationship with EBV infection. Funding acknowledgement: University Grant Council (HK) grants (AoE NPC (AoE/M-06/08); TBRS (T12-401/13R); GRF); University of Hong Kong (CRCG and SRT cancer) Citation Format: WT Lin, L Xia, Dan Dan Wen, CM Tsang, KW Lo, ML Lung, George Sai-Wah Tsao. Establishment and characterization of xenografts and cell lines trom 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 632.

Abstract 4522: Epstein-Barr virus infection suppresses the DNA repair mechanisms in nasopharyngeal epithelial cells via reduction of the H3K4me3 mark

Introduction: Epstein-Barr Virus (EBV) is an oncovirus, which contributes to development of various cancers, including nasopharyngeal carcinoma (NPC). EBV latent proteins play critical roles in modulation host cell histone modifications in order to regulate its signaling pathways. However, the role of EBV in regulation histone modifications in epithelia cell system is still not very clear. Further studies are necessary to characterize the functional role of EBV in regulating epithelial cell modifications. Aim: in this current study, we aim to investigate the role of EBV infection in regulating a promoter and transcription activation histone marker, H3K4me3, in the host cell genome. Methodologies: Chromatin immunoprecipitation sequencing (ChIPseq) was performed by using the next-generation sequencing approach. The ChIP reactions were prepared by utilizing the antibody targeting the H3K4me3 mark in two pairs of immortalized non-tumorigenic nasopharyngeal epithelial (NPE) cell lines, which were artificially infected with (550, 550-EBV, 361, and 361-EBV). The ChIPseq results were validated by the ChIP-QPCR and RT-QPCR. Results: A total of 1747 genes show losses of H3K4me3 in both sets of EBV-infected NPE cell lines. Among them, 628 (36%) genes show losses of H3K4me3 in promoter regions. Interestingly, a total of 18 DNA damage repair signaling members in the base excision repair (BER), homologous recombination, non-homologous end-joining, and the mismatch repair pathways showed significant losses of H3K4me3 in EBV-infected NPE cells. Based on utilizing the DAVID annotation tool for pathway analysis, members in the BER pathway were significantly enriched (FDR = 0.0709, cut-off Conclusion: EBV infection induces changes of host cell H3K4me3 levels and results in down-regulation of the BER members. Acknowledgement: This work was supported by the Research Grants Council of the Hong Kong Special Administrative Region, People9s Republic of China: Grant number AoE/M-06/08 to MLL and the Seed Funding Programme for Basic Research of the University of Hong Kong: Grant number 201308159003 to AKLC. Citation Format: Maria L. Lung, Arthur KL Cheung, Wei Dai, Merrin ML Leong, George SW Tsao. Epstein-Barr virus infection suppresses the DNA repair mechanisms in nasopharyngeal epithelial cells via reduction of the H3K4me3 mark. [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 4522.

Abstract 1280: Germline PALB2 genetic variations of familial esophageal squamous cell carcinoma (ESCC) in Henan, a high risk ESCC region

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background and aims: ESCC is often a deadly cancer diagnosed at late stage with a 5-year survival rate less than 10% in advanced cancers. It is important to elucidate the molecular genetic basis for this deadly cancer to achieve the ultimate goal of early cancer detection and improved clinical management. Our aim is to understand the genetic basis of inherited ESCC by deciphering PALB2 mutation status in high risk northern China. By investigation of PALB2 germline mutations and variants, as compared to healthy individuals, the genetic basis and genomic risk factor associations with inherited EC will be clarified. Methods: Blood was collected from high-risk Henan familial history-positive (FH+) individuals. Blood DNAs were extracted for Sanger sequencing analysis. Results: By Sanger sequencing with primers covering more than 90% of the PALB2 coding sequence, PALB2 variants were detected in exon 4 for 4% (2/50) FH+ ESCC patients from Henan, but no germline protein truncation variations were observed. Since mutations were only observed in exon 4, further mutation screening were confined to exon 4 of PALB2 with an additional 300 FH+ Henan ESCC, 283 FH- Henan ESCC, and 364 Henan non-ESCC control individuals. However, no mutations were detected in the validation samples. Conclusions: Infrequent PALB2 germline mutations were detected in Henan FH+ ESCC patients, suggesting PALB2 may be involved in a small proportion of familial ESCC in Henan. Further study is necessary to clarify if the identified mutations are pathogenic. Citation Format: Josephine Mun Yee Ko, Li Dong Wang, Maria Lung. Germline PALB2 genetic variations of familial esophageal squamous cell carcinoma (ESCC) in Henan, a high risk ESCC region. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1280. doi:10.1158/1538-7445.AM2014-1280

Abstract 1550: Differential angiogenic roles of serum amyloid A 1 (SAA1) isoforms in esophageal squamous cell carcinoma (ESCC)

Esophageal Caner (EC), a highly metastatic and fatal cancer, is ranked the eighth in mortality rate in Hong Kong cancer patients (Hong Kong Cancer Registry, Hospital Authority, 2010). Esophageal Squamous Cell Carcinoma (ESCC) is the predominant type comprising more than 90% of EC. Using a functional complementation approach, SAA1 was identified as one of the tumor suppressor gene candidates. SAA1 is located at chromosome 11p15.1 and is expressed as a secretary protein in liver, human cultured smooth muscle cells, monocyte-macrophage cell lines, and in histologically-normal human epithelial tissues. Genetic polymorphisms of SAA1 have been identified as a risk factor of diseases such as amyloidosis. Three SAA1 isoforms with two single nucleotide polymorphisms at exon three (SAA1.1, 1.3, and 1.5) were observed in the ESCC patients and healthy individuals. The SAA proteins contain the functional YIGSR-like and RGD-like motifs, proteins with these motifs can inhibit angiogenesis, cell adhesion to ECM, growth and metastasis. To understand the anti-tumorigenic and anti-angiogenic roles of the three SAA1 isoforms in ESCC progression, both recombinant proteins and secreted proteins from the conditioned media of lentiviral-infected ESCC cell lines were used for functional assays. For the vascular endothelial cell tube formation assay, the treatments with SAA1.1 and 1.3 proteins showed suppression of tube formation, whereas no significant effects could be observed in the treatment with the SAA1.5. Suppression of cell proliferation and induction of cell death were observed when the endothelial cells were cultured with SAA1.1 and 1.3 proteins. To further elucidate the differential effects among the three SAA1 isoforms in anti-angiogenesis, the cytoskeleton arrangement of the vascular endothelial cells was studied. The SAA1.1 and SAA1.3 proteins could abolish the endothelial cell adhesion by disturbing the formation of stress fibers and focal adhesions. As a conclusion, the present data has shown the variation in anti-angiogenic potential of the three SAA1 isoforms. We acknowledge the financial support of the Food and Health Bureau of the Hong Kong Special Administration Region, China, grant number HMRF 01120886 to HLL. Citation Format: ON YING MAN, Maria Lung, Hong Lok Lung. Differential angiogenic roles of serum amyloid A 1 (SAA1) isoforms in esophageal squamous cell carcinoma (ESCC). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1550. doi:10.1158/1538-7445.AM2014-1550

Abstract 2037: Cyclin D1 overexpression supports stable EBV infection in nasopharyngeal epithelial cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Poorly or undifferentiated nasopharyngeal carcinoma (NPC) is regularly associated with Epstein-Barr virus (EBV) infection. EBV infection of premalignant nasopharyngeal epithelial cells has been postulated to play an important role in the pathogenesis of nasopharyngeal carcinoma. We have previously reported that genetic alterations could be detected in premalignant nasopharyngeal epithelial tissues. It is not clear if these genetic alterations may associate with the establishment of EBV infection. In this study, we have examined the role of cyclin D1 in supporting EBV infection in nasopharyngeal epithelial cells. While cyclin D1 is commonly expressed in nasopharyngeal carcinoma tissues we found that overexpression of cycline D1 is readily detected in dysplastic nasopharyngeal epithelial closely associated with EBV infection. Furthermore, using a panel of immortalized nasopharyngeal epithelial cells, we demonstrated that overexpression of cyclin D1 or CKD4 supports EBV infection and result in the establishment of stable EBV infected nasopharyngeal cell lines. We also observed that EBV infection induced growth inhibition and senescence in nasopharyngeal epithelial cells, which may be the underlying reason for low detection rate of EBV infection in healthy nasopharyngeal epithelial tissues. However, cyclin D1 overexpression suppressed senescence and differentiation in the immortalized nasopharyngeal epithelial cells, suggesting cyclin D1 may play an important role in supporting EBV infection in these cells. We postulated that the altered expression of cyclin D1 or CDK4 in premalignant nasopharyngeal epithelial cells may over-ride the growth suppression effects of EBV infection and support EBV latent infection in premalignant nasopharyngeal epithelial cells. Acknowledgement: This study is sponsored by grant support received from the Hong Kong Research Grant Council (Grant number: GRF780911 and AoE /M-06/08) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2037. doi:1538-7445.AM2012-2037