Tumorigenic Role Research Articles

Mammalian-enabled (MENA) protein enhances oncogenic potential and cancer stem cell-like phenotype in hepatocellular carcinoma cells.

Mammalian‐enabled (MENA) protein is an actin‐regulatory protein that influences cell motility and adhesion. It is known to play a role in tumorigenicity of hepatocellular carcinoma (HCC) but the underlying molecular mechanism remains unknown. This study aimed to investigate the oncogenic potential of MENA and its capacity to regulate cancer stem cell (CSC)‐like phenotypes in HCC cells. Real‐time‐PCR and western blot were used to assess mRNA and protein levels of target genes in human HCC tissue specimens and HCC cell lines, respectively. Stable MENA‐overexpressing HCC cells were generated from HCC cell lines. Transwell cell migration and colony formation assays were employed to evaluate tumorigenicity. Ectopic expression of MENA significantly enhanced cell migration and colony‐forming ability in HCC cells. Overexpression of MENA upregulated several hepatic progenitor/stem cell markers in HCC cells. A high MENA protein level was associated with high mRNA levels of MENA, CD133, cytokeratin 19 (CK19), and epithelial cell adhesion molecule (EpCAM) in human HCC tissues. Overexpression of MENA enhanced epithelial‐to‐mesenchymal transition (EMT) markers, extracellular signal‐regulated kinases (ERK) phosphorylation, and the level of β‐catenin in HCC cells. This study demonstrated that overexpression of MENA in HCC cells promoted stem cell markers, EMT markers, and tumorigenicity. These effects may involve, at least partially, the ERK and β‐catenin signaling pathways.

Sirtuin 6 plays an oncogenic role and induces cell autophagy in esophageal cancer cells

Sirtuin 6, a member of sirtuin family, is generally regarded as a tumor suppressor as it participates in suppressing hypoxia-inducible factor 1α and MYC transcription activity by deacetylating H3K9 (histone H3 lysine 9) and H3K56 (histone H3 lysine) at promoters of target genes, leading to the aerobic glycolysis inhibition and cell growth suppression. However, its expression has recently been reported to be highly elevated in a series of tumors, including prostate cancer, breast cancer, and non-small cell lung cancer, indicating that sirtuin 6 plays dual roles in tumorigenicity in a cell/tumor type-specific manner. To our knowledge, the biological roles of sirtuin 6 in esophageal cancer cells have still been underestimated. In the study, data from quantitative reverse transcriptase polymerase chain reaction-based assays and immunohistochemical assays revealed that sirtuin 6 was remarkably overexpressed in esophageal squamous tumor tissues. Moreover, its upregulation was closely related with clinical features, such as gender, pathology, tumor-node-metastasis, and cell differentiation. Subsequently, the biological tests showed that it promoted cell proliferation and induced the expression of Bcl2, a key anti-apoptotic factor, in esophageal carcinoma cells. Moreover, using the ratio of LC3II/I, a widely recognized autophagy biomarker, we showed that it apparently induced cell autophagy, which was further confirmed by the autophagy flux assays. In addition, results from western blotting assays and immunoprecipitation assays displayed that sirtuin 6 specifically interacted with ULK1 and positively regulated its activity by inhibiting its upstream factor mammalian target of rapamycin activity. In summary, our studies shed insights into the crucial functions of sirtuin 6 in esophageal carcinoma cells and provide evidence supporting sirtuin 6-based personalized therapies in esophageal carcinoma cell patients.

Cell cycle-dependent degradation of the methyltransferase SETD3 attenuates cell proliferation and liver tumorigenesis

Histone modifications, including lysine methylation, are epigenetic marks that influence many biological pathways. Accordingly, many methyltransferases have critical roles in various biological processes, and their dysregulation is often associated with cancer. However, the biological functions and regulation of many methyltransferases are unclear. Here, we report that a human homolog of the methyltransferase SET (SU(var), enhancer of zeste, and trithorax) domain containing 3 (SETD3) is cell cycle-regulated; SETD3 protein levels peaked in S phase and were lowest in M phase. We found that the β-isoform of the tumor suppressor F-box and WD repeat domain containing 7 (FBXW7β) specifically mediates SETD3 degradation. Aligning the SETD3 sequence with those of well known FBXW7 substrates, we identified six potential non-canonical Cdc4 phosphodegrons (CPDs), and one of them, CPD1, is primarily phosphorylated by the kinase glycogen synthase kinase 3 (GSK3β), which is required for FBXW7β-mediated recognition and degradation. Moreover, depletion or inhibition of GSK3β or FBXW7β resulted in elevated SETD3 levels. Mutations of the phosphorylated residues in CPD1 of SETD3 abolished the interaction between FBXW7β and SETD3 and prevented SETD3 degradation. Our data further indicated that SETD3 levels positively correlated with cell proliferation of liver cancer cells and liver tumorigenesis in a xenograft mouse model, and that overexpression of FBXW7β counteracts the SETD3's tumorigenic role. We also show that SETD3 levels correlate with cancer malignancy, indicated by SETD3 levels that the 54 liver tumors are 2-fold higher than those in the relevant adjacent tissues. Collectively, these data elucidated that a GSK3β-FBXW7β-dependent mechanism controls SETD3 protein levels during the cell cycle and attenuates its oncogenic role in liver tumorigenesis.

MB4-2/MB4-3 transcripts of IGH-MMSET fusion gene in t(4;14)pos multiple myeloma indicate poor prognosis.

Multiple myeloma (MM) patients with t(4;14) is a heterogeneous group. Prognostic tools capable of predicting the outcome of patients are currently lacking. The MM SET domain (MMSET) protein is universally overexpressed and has been suggested to have an important tumorigenic role. This study analyzed whether the overexpression of full-length (MB4-1) or truncated forms (MB4-2 and MB4-3) of MMSET influence the prognosis of t(4;14)pos MM patients. A total of 53 symptomatic t(4;14)pos MM patients were retrospectively analyzed. RT-PCR was performed using cDNA from purified CD138+ bone marrow plasma cells to analyze expression and clinical significance of the IGH-MMSET fusion transcripts corresponding to MB4-1, MB4-2 and MB4-3 breakpoints. Among the patients, 25 (47.2%), 12 (22.6%) and 16 (30.2%) had the MB4-1, MB4-2 and MB4-3 breakpoints, respectively. When adjusted to the established prognostic variables including del(17p), ISS stage, serum LDH and serum calcium levels, the pooled MB4-2/MB4-3 subgroup remained a powerful independent adverse factor for PFS (P=0.013) and OS (P=0.029). Bortezomib-based therapy significantly improved the survival of the MB4-1 subgroup but could not overcome the negative effect of the MB4-2/MB4-3 breakpoints. Our results indicate that MB4-2/MB4-3 breakpoints with truncated forms of MMSET define a subset of t(4;14)posMM with poor prognosis.

Diverse expression patterns and tumorigenic role of neurotensin signaling components in colorectal cancer cells.

Colorectal cancer (CRC), which is one of the most common malignancies worldwide, results from an accumulation of genetic and epigenetic modifications including DNA methylation. Neurotensin (NTS), a hormone localized to the gut and central nervous system, mediates its physiological and pathological effects, including growth stimulation for a variety of cancers, through three distinct NTS receptors (NTSRs). Most NTS functions are mediated through the high-affinity receptor NTSR1, and expression of NTSR1 is increased in many cancers including CRC. In this study, we investigated the expression profiles and cellular functions of the NTSRs, especially NTSR1, in CRC cells. We showed that expression levels for NTS and NTSR1 varied, that NTSR2 expression was not detectable and that NTSR3 was consistently expressed in all CRC cell lines examined. Treatment with the demethylating agent, 5-aza-2′-deoxycytidine, augmented levels of NTSR1/2 in Caco2 and DLD1 cells, which have little or no transcripts for NTSR1/2 suggesting that DNA methylation suppresses NTSR1/2 expression. In addition, we demonstrated that knockdown of NTSR1 decreased cell growth and migration in HCT116 and HT29 cells. Finally, we showed that treatment with SR48692, an antagonist of NTSR1, also inhibited cell proliferation and migration in the CRC cells. Our findings identify promoter methylation as an important process regulating the differential expression or silencing of NTSR1/2 in CRC cells. Moreover, inhibition of NTSR1 repressed tumorigenic effects in CRC cells, suggesting that NTSR1 may be used as a therapeutic target for CRC.

Modeling Developmental and Tumorigenic Aspects of Trilateral Retinoblastoma via Human Embryonic Stem Cells.

SummaryHuman embryonic stem cells (hESCs) provide a platform for studying human development and understanding mechanisms underlying diseases. Retinoblastoma-1 (RB1) is a key regulator of cell cycling, of which biallelic inactivation initiates retinoblastoma, the most common congenital intraocular malignancy. We developed a model to study the role of RB1 in early development and tumor formation by generating RB1-null hESCs using CRISPR/Cas9. RB1−/− hESCs initiated extremely large teratomas, with neural expansions similar to those of trilateral retinoblastoma tumors, in which retinoblastoma is accompanied by intracranial neural tumors. Teratoma analysis further revealed a role for the transcription factor ZEB1 in RB1-mediated ectoderm differentiation. Furthermore, RB1−/− cells displayed mitochondrial dysfunction similar to poorly differentiated retinoblastomas. Screening more than 100 chemotherapies revealed an RB1–/–-specific cell sensitivity to carboplatin, exploiting their mitochondrial dysfunction. Together, our work provides a human pluripotent cell model for retinoblastoma and sheds light on developmental and tumorigenic roles of RB1.

Sox2 is dispensable for primary melanoma and metastasis formation.

Tumor initiation and metastasis formation in many cancers have been associated with emergence of a gene expression program normally active in embryonic or organ-specific stem cells. In particular, the stem cell transcription factor Sox2 is not only expressed in a variety of tumors, but is also required for their formation. Melanoma, the most aggressive skin tumor, derives from melanocytes that during development originate from neural crest stem cells. While neural crest stem cells do not express Sox2, expression of this transcription factor has been reported in melanoma. However, the role of Sox2 in melanoma is controversial. To study the requirement of Sox2 for melanoma formation, we therefore performed CRISPR-Cas9-mediated gene inactivation in human melanoma cells. In addition, we conditionally inactivated Sox2 in a genetically engineered mouse model, in which melanoma spontaneously develops in the context of an intact stroma and immune system. Surprisingly, in both models, loss of Sox2 did neither affect melanoma initiation, nor growth, nor metastasis formation. The lack of a tumorigenic role of Sox2 in melanoma might reflect a distinct stem cell program active in neural crest stem cells and during melanoma formation.

Morphological characterization of colorectal cancers in The Cancer Genome Atlas reveals distinct morphology–molecular associations: clinical and biological implications

The Cancer Genome Atlas data on colorectal carcinoma have provided a comprehensive view of the tumor's genomic alterations and their tumorigenic roles. Tumor morphology, however, has not been fully integrated into the analysis. The aim of this study was to explore relevant associations between tumor morphology and the newly characterized genomic alterations in colorectal carcinoma. Two hundred and seven colorectal carcinomas that had undergone whole-exome sequencing as part of The Cancer Genome Atlas project and had adequate virtual images in the cBioPortal for Cancer Genomics constituted our study population. Upon analysis, a tight association between ‘microsatellite instability-high histology' and microsatellite instability-high (P<0.001) was readily detected and helped validate our image-based histology evaluation. Further, we showed, (1) among all histologies, the not otherwise specified type had the lowest overall mutation count (P<0.001 for entire cohort, P<0.03 for the microsatellite-instable group), and among the microsatellite-instable tumors, this type also correlated with fewer frameshift mutations in coding mononucleotide repeats of a defined set of relevant genes (P<0.01); (2) cytosine phosphate guanine island methylator phenotype-high colorectal cancers with or without microsatellite instability tended to have different histological patterns: the former more often mucinous and the latter more often not otherwise specified; (3) mucinous histology was associated with more frequent alterations in BRAF, PIK3CA, and the transforming growth factor-β pathway when compared with non-mucinous histologies (P<0.001, P=0.01, and P<0.001, respectively); and (4) few colorectal cancers (<9%) exhibited upregulation of immune-inhibitory genes including major immune checkpoints; these tumors were primarily microsatellite-instable (up to 43%, vs <3% in microsatellite-stable group) and had distinctly non-mucinous histologies with a solid growth. These morphology–molecular associations are interesting and propose important clinical implications. The morphological patterns associated with alterations of immune checkpoint genes bear the potential to guide patient selection for clinical trials that target immune checkpoints in colorectal cancer, and provide directions for future studies.

Recurrent fusion RNA DUS4L-BCAP29 in non-cancer human tissues and cells.

Traditional gene fusions are involved in the development of various neoplasia. DUS4L-BCAP29, a chimeric fusion RNA, has been reported to be a cancer-fusion in prostate and gastric cancer, in addition to playing a tumorigenic role. Here, we showed that the DUS4L-BCAP29 fusion transcript exists in a variety of normal tissues. It is also present in non-cancer epithelial, as well as in fibroblast cell lines. Quantitatively, the fusion transcript has a comparable expression in non-cancerous, gastric and prostate cell lines and tissues as in the cancer cell lines and tissues. The loss-of-function approach as previously reported is not sufficient to prove the functionality of the fusion. On the other hand, the gain-of-function approach showed that overexpression of DUS4L-BCAP29 promotes cell growth and motility, even in non-cancer cells. Finally, we provide further evidence that the fusion transcript is a product of cis-splicing between adjacent genes. In summary, we believe that in contrast to traditional gene fusions, DUS4L-BCAP29 cannot be used as a cancer biomarker. Instead, it is a fusion transcript that exists in normal physiology and that its pro-growth effect is not unique to cancer cells.

ALG2 regulates glioblastoma cell proliferation, migration and tumorigenicity

Apoptosis-linked gene-2 (ALG-2), also known as programmed cell death 6 (PDCD6), has recently been reported to be aberrantly expressed in various tumors and required for tumor cell viability. The aim of the present study was to investigate whether ALG-2 plays a crucial role in tumor cell proliferation, migration and tumorigenicity. In this study, we examined the expression of PDCD6 in glioblastoma cell lines and found that ALG-2 was generally expressed in glioblastoma cell lines. We also performed an analysis of an online database and found that high expression of ALG-2 was associated with poor prognosis (p = 0.039). We found that over-expression of ALG2 in glioblastoma could inhibit cell proliferation and, conversely, that down-regulation of ALG2 could promote cell proliferation. Further studies showed that over-expression of ALG2 inhibited the migration of tumor cells, whereas down-regulation of ALG2 promoted tumor cell migration. Finally, in vitro and in vivo studies showed that over-expression of ALG2 inhibited the tumorigenic ability of tumor cells, while down-regulation of ALG2 promoted tumor cell tumorigenic ability. In conclusion, ALG2 has a tumor suppressive role in glioblastoma and might be a potential target for the treatment of glioblastoma.

Myeloid cells as a target for oligonucleotide therapeutics: turning obstacles into opportunities.

Immunotherapies emerged as an alternative for cancer treatment, yet their clinical efficacies are still limited, especially in case of solid tumors. Myeloid immune cells, such as macrophages and myeloid-derived suppressor cells (MDSCs), are often hijacked by tumors and become pivotal inhibitors of antitumor immunity. Immunosuppressive functions of tumor-associated myeloid cells result from the activity of Signal Transducer and Activator of Transcription 3 (STAT3), a transcription factor with well-defined tumorigenic and tolerogenic roles in human cancers. To overcome challenges in the development of pharmacological STAT3 inhibitors, we recently developed oligonucleotide-based strategies for cell-selective, in vivo STAT3 targeting. Conjugation of a STAT3siRNA or decoy STAT3 inhibitors to synthetic Toll-like Receptor 9 (TLR9) agonists, CpG oligonucleotides, allowed for selective delivery into TLR9-positive cells. Cellular target for CpG-STAT3 inhibitors include non-malignant, tumor-associated myeloid cells, such as polymorphonuclear MDSCs, as well as cancer cells in acute myeloid leukemia, B cell lymphoma and in certain solid tumors. The chemically modified CpG-STAT3 inhibitors resist serum nucleases and thus can be administered intravenously. Their potency relies on the intracellular gain-of-function effect: release of the central immune checkpoint regulator (STAT3) to unleash proinflammatory signaling (CpG/TLR9) in the same antigen-presenting cell. At the cellular level, CpG-STAT3 inhibitors exert two-pronged effect by rescuing T cells from the immune checkpoint control while decreasing survival of cancer cells. In this article, we review the preclinical data on CpG-STAT3 inhibitors and discuss perspectives of using TLR9-targeted delivery of oligonucleotide therapeutics for the generation of novel, more effective and safer cancer immunotherapies.

Doublecortin-like kinase 1 is a novel biomarker for prognosis and regulates growth and metastasis in basal-like breast cancer

Doublecortin like kinase 1 (DCLK1) was observed overexpressed in several types of human cancers, and played a functional role in tumorigenicity, but little is known about the biological function of DCLK1 in basal-like breast cancer. In our results, the expressions of DCLK1 mRNA and protein were elevated in basal-like breast cancer tissues and cell lines. DCLK1 high-expression associated with clinical stage, lymph node metastasis, distant metastasis and histological grade. DCLK1 high-expression was an independent unfavorable prognostic factor for basal-like breast cancer patients. The biological experiments suggested knockdown of DCLK1 induced inhibition of basal-like breast cancer cells proliferation, migration and invasion. In conclusion, DCLK1 serves as a prognostic biomarker for basal-like breast cancer and a potential therapeutic target for basal-like breast cancer.

Autotaxin-lysophosphatidic acid receptor signalling regulates hepatitis C virus replication

Chronic hepatitis C is a global health problem with an estimated 170 million hepatitis C virus (HCV) infected individuals at risk of progressive liver disease and hepatocellular carcinoma (HCC). Autotaxin (ATX, gene name: ENPP2) is a phospholipase with diverse roles in the physiological and pathological processes including inflammation and oncogenesis. Clinical studies have reported increased ATX expression in chronic hepatitis C, however, the pathways regulating ATX and its role in the viral life cycle are not well understood. In vitro hepatocyte and ex vivo liver culture systems along with chimeric humanized liver mice and HCC tissue enabled us to assess the interplay between ATX and the HCV life cycle. HCV infection increased hepatocellular ATX RNA and protein expression. HCV infection stabilizes hypoxia inducible factors (HIFs) and we investigated a role for these transcription factors to regulate ATX. In vitro studies show that low oxygen increases hepatocellular ATX expression and transcriptome analysis showed a positive correlation between ATX mRNA levels and hypoxia gene score in HCC tumour tissue associated with HCV and other aetiologies. Importantly, inhibiting ATX-lysophosphatidic acid (LPA) signalling reduced HCV replication, demonstrating a positive role for this phospholipase in the viral life cycle. LPA activates phosphoinositide-3-kinase that stabilizes HIF-1α and inhibiting the HIF signalling pathway abrogates the pro-viral activity of LPA. Our data support a model where HCV infection increases ATX expression which supports viral replication and HCC progression. Chronic hepatitis C is a global health problem with infected individuals at risk of developing liver disease that can progress to hepatocellular carcinoma. Autotaxin generates the biologically active lipid lysophosphatidic acid that has been reported to play a tumorigenic role in a wide number of cancers. In this study we show that hepatitis C virus infection increases autotaxin expression via hypoxia inducible transcription factor and provides an environment in the liver that promotes fibrosis and liver injury. Importantly, we show a new role for lysophosphatidic acid in positively regulating hepatitis C virus replication.

Integrin β4 promotes cell invasion and epithelial-mesenchymal transition through the modulation of Slug expression in hepatocellular carcinoma

Integrin β4 (ITGB4) is a transmembrane receptor involved in tumorigenesis and the invasiveness of many cancers. However, its role in hepatocellular carcinoma (HCC), one of the most prevalent human cancers worldwide, remains unclear. Here, we examined the involvement of ITGB4 in HCC and explored the underlying mechanisms. Real-time PCR and immunohistochemical analyses of tissues from 82 patients with HCC and four HCC cell lines showed higher ITGB4 levels in tumor than in adjacent non-tumor tissues and in HCC than in normal hepatic cells. Silencing of ITGB4 repressed cell proliferation, colony forming ability and cell invasiveness, whereas ectopic expression of ITGB4 promoted the proliferation and invasion of HCC cells and induced epithelial to mesenchymal transition (EMT) in parallel with the upregulation of Slug, as shown by transwell assays, WB and immunocytochemistry. Knockdown of Slug reduced cell viability inhibited invasion and reversed the effects of ITBG4 overexpression on promoting EMT, and AKT/Sox2-Nanog may also be involved. In a xenograft tumor model induced by injection of ITGB4-overexpressing cells into nude mice, ITGB4 promoted tumor growth and metastasis to the lungs. Taken together, our results indicate that ITGB4 plays a tumorigenic and pro-metastatic role mediated by Slug and suggest IGTB4 could be a prognostic indicator or a therapeutic target in patients with HCC.

WIF-1 gene inhibition and Wnt signal transduction pathway activation in NSCLC tumorigenesis

The aim of the present study is to explore the differential expression of key molecules associated with Wnt signaling in both clinical non-small cell lung cancer (NSCLC) tissue and adjacent normal lung tissue, and to discuss the tumorigenic role of the activation of Wnt signaling pathways in NSCLC. A total of 52 NSCLC patients were employed in the present study. Lung cancer tissue samples and paracarcinoma tissue samples were obtained from these patients, who had undergone surgical resection of their primary cancer. The cases were diagnosed by hematoxylin and eosin staining. Using reverse transcription-quantitative polymerase chain reaction and immunohistochemical straining, the messenger RNA (mRNA) and protein expression levels of Wnt inhibitory factor-1 (WIF-1) and important molecules associated with Wnt signaling pathways were detected. Compared with normal tissues, a marked decreased in the mRNA and protein expression levels of WIF-1, and an increase in β-catenin and cyclin D1 expression, were observed in tumor tissues. This suggests that the activation of the Wnt/β-catenin signaling pathway may be closely associated with lymph nodal metastasis and lower pathological classification. However, no obvious difference could be observed in adenomatous polyposis coli (APC) expression levels between lung cancer tissues and adjacent tissues to the carcinoma. The activation of the Wnt/β-catenin signaling pathway in NSCLC could be initiated by WIF-1 gene inhibition without APC expression changes, and this may be different to the mechanism in other tumors.

MA16.01 Targeted Gene Therapy for Tobacco Carcinogen-Induced Lung Cancer

Rab25, a member of Rab family of small GTPases, is associated with progression of various types of human cancer including lung cancer that is the leading cause of cancer-associated deaths around the globe. In this study, we report the gene therapeutic effect of short hairpin Rab25 (shRab25) on 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorgenesis in female A/J mice. Initially, Mice (six-week-old) were injected with single dose of NNK (2 mg/0.1 ml saline/mouse) by intraperitoneal injection to induce the tumor. 8 weeks later, shRab25 was delivered with GPT-SPE (glycerol propoxylate triacrylate (GPT) and spermine) complex into tobacco-induced lung cancer models through a nose-only inhalation system twice a week for 2 month. Remarkably, aerosol-delivered shRab25 significantly decreased the expression level of Rab25 and other prominent apoptosis related proteins in female A/J mice. The apoptosis in these mice were determined by detecting the expression level of Bcl-2, PCNA, Bax and further confirmed by TUNEL assay. Our results strongly confirm the tumorigenic role of Rab25 in tobacco carcinogen induced-lung cancer and hence demonstrate aerosol delivery of shRab25 as a therapeutic target for lung cancer treatment.

Spatiotemporal Patterns of RING1 Expression after Rat Spinal Cord Injury

Ring finger protein 1 (RING1) is a RING domain characterized protein belonging to the RING finger family. It is an E3 ubiquitin-protein ligase that mediated monoubiquitination of histone H2A and the core component of PRC1 complex, which is the repressive multiprotein complex of Polycomb group (PcG). Previous studies showed the important tumorigenic role of RING1 via promoting cell proliferation and the crucial function in maintaining transcriptional program stability during development. However, its mechanism for spinal cord injury (SCI) is still unknown. In our research, we established an acute SCI model in adult rats and studied the expression and function profiles of RING1. RING1 protein level detected by western blot peaked at day 3 after trauma and then decreased gradually. Immunohistochemistry showed the increase of RING1 expression displayed in the white matter more obviously than in the gray matter. Furthermore, increased co-expression of RING1 and GFAP confirmed activated astrocytes in injured spinal cord via double immunofluorescence staining. Meanwhile, we also found the co-localization of PCNA, a famous marker of proliferative cells, with RING1 and GFAP, which indicated RING1 might play a role in astrocyte proliferation after SCI. In vitro studies, RING1 protein level in C6 cells increased after LPS challenge and RING1 was required for astrocyte proliferation and activation induced by LPS. In summary, we took a new insight into the function of RING1 in the cellular and molecular mechanism underlying the pathophysiology of SCI.

Genome-Scale ORF Screen for Mediators of NF-κb Activation in DLBCL

Cancer genome and transcriptome sequencing can identify novel oncogenes and tumor suppressors, discover distinct cancer subtypes, and predict therapeutic responses. Analysis of the coding genome of diffuse large B cell lymphoma (DLBCL) has identified various genetic mutations in the ABC and GCB molecular subtypes. Notably, ABC DLBCLs have recurrent activating mutations involving the canonical NF-kB pathway. Although an oncogenic role for constitutive NF-kB activity has been demonstrated in ABC DLBCL, the molecular mechanisms of various oncogenic mutations are still elusive. To understand the pathogenesis of DLBCL, we used high-throughput exome and transcriptome sequencing of more than 500 DLBCL biopsies.To enable functional screening of mutant alleles identified by exome-seq and RNA-seq, we are constructing an inducible retroviral expression library of over 200 open reading frames (ORFs) of genes mutated in DLBCL. We have cloned the mutant and wildtype forms of these genes together with a unique 26-base-pair 'bar code' to facilitate screening with high-throughput sequencing.We have transduced a subset of this ORF library into DLBCL cell lines, induced ORF expression by doxycycline, and FACS sorted into high and low populations based on expression the known NF-κB target gene CD83. We then performed barcode sequencing to assay relative enrichment of ORFs that confer higher or lower NF-κB activity. ORF screening revealed that MYD88L265P, CARD11L232LI, both well-characterized gain-of-function mutants, were among the highest ranking genes for induction of NF-κB activity. Interestingly, we observed enrichment for USP7D271E in NF-κB high populations, though these genes have not been shown to have a tumorigenic role in DLBCL.Ubiquitin Specific Protease-7 (USP7) is a regulator of NF-κB transcriptional activity, in part by de-ubiquitinating p65. To test how USP7 mutations contribute to NF-κB activity, we engineered the ABC DLBCL cell line TMD8 to express an NF-κB reporter consisting of the NF-κB transcriptional response element (TRE) fused to GFP. NF-κB activity upon overexpression of wildtype or mutant USP7 was monitored by flow cytometry (FACS). We observed that the mutated form of USP7 significantly increased NF-κB activity over that seen with wildtype, suggesting this is a gain-of-function mutant.We next used the CRISPR gene-targeting system to probe the function of USP7 genetically. Single guide RNAs (sgRNAs) targeting the USP7 coding regions were coexpressed in TMD8 cells with the endonuclease Cas9. SgRNA-expressing, GFP+ cells were monitored over time among total live cells by flow cytometry, and a relative reduction of the GFP+ population was observed in the sgUSP7 population. These results suggest that USP7 may function as an oncogene in DLBCL.This ongoing work demonstrates the efficacy of a high-throughput ORF expression screen to characterize mutations found in the genomic landscape of the DLBCL. DisclosuresNo relevant conflicts of interest to declare.

Expression of Epstein-Barr virus among oral potentially malignant disorders and oral squamous cell carcinomas in the South Indian tobacco-chewing population.

Oral cancer is the sixth most common malignancy in the world. Viruses are the causative agents of approximately 10-15% of all cancers worldwide (Cancers, 6, 2014 and 2155). The tumorigenic roles of Epstein-Barr virus in oral cancer are unclear. Literature search results are conflicting and dependent on various factors such as geographical/regional variations, sociocultural lifestyles, dietary habits, chewing/smoking tobacco habit. This study is the first original observation about frequency of Epstein-Barr virus among South Indian tobacco-chewing patients to elucidate its involvement in oral carcinogenesis and to know whether this can be a valuable diagnostic and prognostic indicator. A total number of 75 tobacco chewer subjects aged between 23 and 76 years with histopathologically confirmed oral potentially malignant disorders (25), oral squamous cell carcinoma (25), and age-matched healthy controls (25) formed the study group. Immunohistochemical expression of Epstein-Barr virus latent membrane protein 1 was assessed among cases and healthy controls. Out of the total 75 subjects, six subjects (8%) were positive for Epstein-Barr virus antigen and 69 subjects (92%) negative. The antigen positivity was observed among two cases of moderately differentiated oral squamous cell carcinoma, two cases of leukoplakia, and two healthy controls. No significant association between Epstein-Barr virus positivity was observed among oral potentially malignant disorders and oral squamous cell carcinoma among South Indian tobacco-chewing patients. This can be partially explained by the methodology employed, by the patient population analyzed and different habits in various geographical regions.

Suppression of Tobacco Carcinogen-Induced Lung Tumorigenesis by Aerosol-Delivered Glycerol Propoxylate Triacrylate-Spermine Copolymer/Short Hairpin Rab25 RNA Complexes in Female A/J Mice.

Rab25, a member of Rab family of small guanosine triphosphatase, is associated with progression of various types of human cancers, including lung cancer, the leading cause of cancer-associated deaths around the globe. In this study, we report the gene therapeutic effect of short hairpin Rab25 RNA (shRab25) on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorigenesis in female A/J mice. Initially, mice (6 weeks old) were injected with single dose of NNK (2 mg/0.1 mL saline/mouse) by intraperitoneal injection to induce the tumor. Eight weeks later, shRab25 was complexed with glycerol propoxylate triacrylate-spermine (GPT-SPE) copolymer and delivered into tobacco-induced lung cancer models through a nose-only inhalation system twice a week for 2 months. GPT-SPE/shRab25 largely decreased the tobacco-induced tumor numbers and tumor volume in the lungs compared to GPT-SPE- or GPT-SPE/shScr-delivered groups. Remarkably, aerosol-delivered GPT-SPE/shRab25 significantly decreased the expression level of Rab25 and other prominent apoptosis-related proteins in female A/J mice. The apoptosis in these mice was determined by detecting the expression level of Bcl-2, proliferating cell nuclear antigen, Bax, and further confirmed by TUNEL assay. Our results strongly confirm the tumorigenic role of Rab25 in tobacco carcinogen-induced lung cancer and hence demonstrate aerosol delivery of shRab25 as a therapeutic target for lung cancer treatment.