Tumor Suppressor Gene Function Research Articles

The tumor suppressor gene RBM5 inhibits lung adenocarcinoma cell growth and induces apoptosis

BackgroundThe loss of tumor suppressor gene (TSG) function is a critical step in the pathogenesis of human lung cancer. RBM5 (RNA-binding motif protein 5, also named H37/LUCA-15) gene from chromosome 3p21.3 demonstrated tumor suppressor activity. However, the role of RBM5 played in the occurrence and development of lung cancer is still not well understood.MethodPaired non-tumor and tumor tissues were obtained from 30 adenocarcinomas. The expression of RBM5 mRNA and protein was examined by RT-PCR and Western blot. A549 cell line was used to determine the apoptotic function of RBM5 in vitro. A549 cells were transiently transfected with pcDNA3.1-RBM5. AnnexinV analysis was performed by flow cytometry. Expression of Bcl-2, cleaved caspase-3, caspase-9 and PAPP proteins in A549 lung cancer cells and the A549 xenograft BALB/c nude mice model was determined by Western blot. Tumor suppressor activity of RBM5 was also examined in the A549 xenograft model treated with pcDNA3.1-RBM5 plasmid carried by attenuated Salmonella typhi Ty21a.ResultThe expression of RBM5 mRNA and protein was decreased significantly in adenocarcinoma tissues compared to that in the non-tumor tissues. In addition, as compared to the vector control, a significant growth inhibition of A549 lung cancer cells was observed when transfected with pcDNA3.1-RBM5 as determined by cell proliferation assay. We also found that overexpression of RBM5 induced both early and late apoptosis in A549 cells using AnnexinV/PI staining as determined by flow cytometry. Furthermore, the expression of Bcl-2 protein was decreased, whereas the expression of cleaved caspase-3, caspase-9 and PARP proteins was significantly increased in the RBM5 transfected cells; similarly, expression of decreased Bcl-2 and increased cleaved caspase-3 proteins was also examined in the A549 xenograft model. More importantly, we showed that accumulative and stable overexpression of RBM5 in the A549 xenograft BALB/c nude mice model significantly inhibited the tumor growth rate in vivo as compared to that in the control.ConclusionOur study demonstrates that RBM5 can inhibit the growth of lung cancer cells and induce apoptosis both in vitro and in vivo, which suggests that RBM5 might be used as a potential biomarker or target for lung cancer diagnosis and chemotherapy. Moreover, we propose a novel animal model set up in BALB/c nude mice treated with attenuated Salmonella as a vector carrying plasmids to determine RBM5 function in vivo.

Abstract B41: Thymoquinone induces-cell cycle non-specific-cell death in cisplatin-resistant ovarian cancer cell through up-regulation of PTEN expression

Abstract Ovarian carcinoma is one of the most prevalent and lethal gynecological malignancies, and is the fifth leading cause of cancer death among women in the United Stat. Like most malignancies, ovarian cancer is a heterogeneous disease characterized by multiple molecular alterations. These include loss of function of tumor suppressor genes, deregulation of Bcl-2 family proteins, inhibition of caspase activation, and activation of cellular survival signaling pathways, such as HER-2/neu and phosphatidylinositol-3 kinase PI3K/Akt. Over years the use of natural compounds as anticancer agents acquired great importance. Thymoquinone (TQ) is the main bioactive component of the volatile oil of the black seed (Nigella sativa, Ranunclulaceae family). TQ has been used as antioxidant, anti-inflammatory, and antineoplastic medicines for more than 2,000 years. Previous studies reported that TQ exhibited inhibitory effects on cell proliferation of many types of cancer cell lines, including breast adenocarcinoma, lung adenocarcinoma, colorectal cancer, etc. Here, we report that TQ greatly inhibited cell proliferation of cisplatin-resistant ovarian cancer cells (A2780/CP70) 24, 48 and 72 hours after treatment in a concentration-dependent manner. However, our flow cytometry data revealed that TQ is a cell cycle non-specific for CP70 cells. Our results also showed that TQ upregulated PTEN and p53 in CP70 cells while downregulated phospho-Akt in a dose-dependent manner. Current investigations are ongoing to fully understand the mechanism of action of TQ in cisplatin-resistant ovarian cancer cells.

Prdx1 deficiency in mice promotes tissue specific loss of heterozygosity mediated by deficiency in DNA repair and increased oxidative stress

The loss of the H2O2 scavenger protein encoded by Prdx1 in mice leads to an elevation of reactive oxygen species (ROS) and tumorigenesis of different tissues. Loss of heterozygosity (LOH) mutations could initiate tumorigenesis through loss of tumor suppressor gene function in heterozygous somatic cells. A connection between the severity of ROS and the frequency of LOH mutations in vivo has not been established. Therefore, in this study, we characterized in vivo LOH in ear fibroblasts and splenic T cells of 3–4 month old Prdx1 deficient mice. We found that the loss of Prdx1 significantly elevates ROS amounts in T cells and fibroblasts. The basal amounts of ROS were higher in fibroblasts than in T cells, probably due to a less robust Prdx1 peroxidase activity in the former. Using Aprt as a LOH reporter, we observed an elevation in LOH mutation frequency in fibroblasts, but not in T cells, of Prdx1−/− mice compared to Prdx1+/+ mice. The majority of the LOH mutations in both cell types were derived from mitotic recombination (MR) events. Interestingly, Mlh1, which is known to suppress MR between divergent sequences, was found to be significantly down-regulated in fibroblasts of Prdx1−/− mice. Therefore, the combination of elevated ROS amounts and down-regulation of Mlh1 may have contributed to the elevation of MR in fibroblasts of Prdx1−/− mice. We conclude that each tissue may have a distinct mechanism through which Prdx1 deficiency promotes tumorigenesis.

Abstract LB-104: Prostate-specific inactivation of Atbf1 causes mouse prostatic intraepithelial neoplasia

Abstract Cancer initiation and progression is triggered by loss of function of tumor suppressor genes. Previously, we have identified the AT-motif Binding Factor 1 (ATBF1) as a candidate tumor suppressor gene at 16q22 in the prostate. It undergoes frequent somatic mutations, reduced expression, or mislocalization in human cancers. However, its tumor suppressor role has not been examined in vivo. In the current study, we generated a conditional Atbf1 knockout mouse line using the Cre-loxP system. Tissue-specific inactivation of Atbf1 in prostatic epithelial cells caused hyperplasia in the dorsal lobes at as early as 4 months of age, which further progressed to mouse prostatic intraepithelial neoplasia (mPIN) in all four lobes. These precancerous lesions harbored molecular aspects similar to human PIN and prostate cancer, including increased cell proliferation, attenuated the basal cell layer, loss of the cell junction protein E-cadherin, and loss of an intact smooth muscle layer. Meanwhile, we observed elevated levels of Muc1 and p-Erk 1/2 in mPIN lesions. In addition, we identified and validated a number of genes that were disregulated in the Atbf1 knockout mice, and many of those genes encode secretory proteins. Supportively, an altered secretory protein profile was observed in the dorsal lobes. Taken together, our data demonstrated that loss of Atbf1 caused precancerous lesions in the prostate, thus providing in vivo evidence to a tumor suppressor role of the ATBF1 gene. 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 LB-104. doi:1538-7445.AM2012-LB-104

Abstract LB-259: Knockdown of slingshot2 serine phosphatase induces caspase3 activation in human carcinoma cell lines with the loss of the Birt-Hogg-Dubé tumor suppressor gene

Abstract Birt-Hogg-Dubé (BHD) Syndrome, is a dominantly familial cancer syndrome associated with susceptibility to renal cancer carcinoma (RCC) caused by inactivating mutations in the folliculin (FLCN) gene. The precise functions of the FLCN gene product are still under investigation but RCC from BHD patients show loss of the wild type allele consistent with a tumour suppressor gene function. In search for potential synthetic-lethal targets for FLCN using a phosphatase siRNA library screen approach, we found that knockdown of SSH2 serine phosphatase (one of the three members of Slingshot family and actively involved in Actin reorganization) specifically induced caspase 3/7 activity in a dose-dependent manner (up to 6 fold increase, 10nM, 72hrs) in two human cell lines (BHD-origin renal cell carcinoma UOK-257 and thyroid carcinoma FTC-133) with loss of FLCN expression, but not in their folliculin expressing isogenic cell lines. SSH2 siRNA-induced knockdown was verified by real-time PCR (up to 60% reduction in SSH2 transcripts, 10nM siRNA, 48hrs), but SSH1 and SSH3 transcripts were increased up to 3 fold (suggesting a compensatory regulatory mechanism among members of SSH family). Further investigation of the underlining mechanism of caspase induction by SSH2 knockdown in FLCN-null cells exhibited that there was a dose-dependent increase in G1 cell population (up to 20% increase, 20nM SSH2 siRNA, 72hrs). Combination treatment of SSH2 + SSH1 siRNAs, but not SSH2 + SSH3 siRNAs potentiated induction of caspase 3 activity and G1 cell cycle arrest. These data indicate that apoptotic cell death in FLCN-null cells can be triggered by SSH2 knockdown through G1 arrest and provide a tool to develop potential therapeutic agents for BHD patients. 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 LB-259. doi:1538-7445.AM2012-LB-259

Cooperation between Rb and Arf in suppressing mouse retinoblastoma

Retinoblastoma is a pediatric cancer that has served as a paradigm for tumor suppressor gene function. Retinoblastoma is initiated by RB gene mutations, but the subsequent cooperating mutational events leading to tumorigenesis are poorly characterized. We investigated what these additional genomic alterations might be using human retinoblastoma samples and mouse models. Array-based comparative genomic hybridization studies revealed deletions in the CDKN2A locus that include ARF and P16INK4A, both of which encode tumor suppressor proteins, in both human and mouse retinoblastoma. Through mouse genetic analyses, we found that Arf was the critical tumor suppressor gene in the deleted region. In mice, inactivation of one allele of Arf cooperated with Rb and p107 loss to rapidly accelerate retinoblastoma, with frequent loss of heterozygosity (LOH) at the Arf locus. Arf has been reported to exhibit p53-independent tumor suppressor roles in other systems; however, our results showed no additive effect of p53 and Arf coinactivation in promoting retinoblastoma. Moreover, p53 inactivation completely eliminated any selection for Arf LOH. Thus, our data reveal important insights into the p53 pathway in retinoblastoma and show that Arf is a key collaborator with Rb in retinoblastoma suppression.

Generation of Dhx9‐deficient clones in T‐cell development with a mitotic recombination technique

Mitotic recombination is an effective tool for generating mutant clones in somatic tissues. Because of difficulties associated with detecting and quantifying mutant clones in mice, this technique is limited to analysis of growth-related phenotypes induced by loss function of tumor suppressor genes. Here, we used the polymorphic CD45.1/CD45.2 alleles on chromosome 1 as pan-hematopoietic markers to track mosaic clones generated through mitotic recombination in developing T cells. We show that lineage-specific mitotic recombination can be induced and reliably detected as CD45.1 or CD45.2 homozygous clones from the CD45.1/CD45.2 heterozygous background. We have applied this system in the analysis of a lethal mutation in the Dhx9 gene. Mosaic analysis revealed a stage-specific role for Dhx9 during T-cell maturation. Thus, the experimental system described in this study offers a practical means for mosaic analysis of germline mutations in the hematopoietic system.

Zinc-finger protein 331, a novel putative tumor suppressor, suppresses growth and invasiveness of gastric cancer

Zinc-finger protein 331 (ZNF331), a Kruppel-associated box zinc-finger protein gene, was identified as a putative tumor suppressor in our previous study. However, the role of ZNF331 in tumorigenesis remains elusive. We aimed to clarify its epigenetic regulation and biological functions in gastric cancer. ZNF331 was silenced or downregulated in 71% (12/17) gastric cancer cell lines. A significant downregulation was also detected in paired gastric tumors compared with adjacent non-cancer tissues. In contrast, ZNF331 was readily expressed in various normal adult tissues. The downregulation of ZNF331 was closely linked to the promoter hypermethylation as evidenced by methylation-specific PCR, bisulfite genomic sequencing and reexpression by demethylation agent treatment. DNA sequencing showed no genetic mutation/deletion of ZNF331 in gastric cancer cell lines. Ectopic expression of ZNF331 in the silenced cancer cell lines MKN28 and HCT116 significantly reduced colony formation and cell viability, induced cell cycle arrests and repressed cell migration and invasive ability. Concordantly, knockdown of ZNF331 increased cell viability and colony formation ability of gastric cancer cell line MKN45. Two-dimensional gel electrophoresis and mass spectrometry-based comparative proteomic approach were applied to analyze the molecular basis of the biological functions of ZNF331. In all, 10 downstream targets of ZNF331 were identified to be associated with regulation of cell growth and metastasis. The tumor-suppressive effect of ZNF331 is mediated at least by downregulation of genes involved in cell growth promotion (DSTN, EIF5A, GARS, DDX5, STAM, UQCRFS1 and SET) and migration/invasion (DSTN and ACTR3), and upregulation of genome-stability gene (SSBP1) and cellular senescence gene (PNPT1). A novel target of ZNF331 (DSTN) was functionally validated. Overexpression of DSTN in BGC-823 cells increased colony formation and migration ability. In conclusion, our results suggest that ZNF331 possesses important functions for the suppression of gastric carcinogenesis as a novel functional tumor-suppressor gene.

Long-range epigenetic silencing of chromosome 5q31 protocadherins is involved in early and late stages of colorectal tumorigenesis through modulation of oncogenic pathways.

Loss of tumour suppressor gene function can occur as a result of epigenetic silencing of large chromosomal regions, referred to as long-range epigenetic silencing (LRES), and genome-wide analyses have revealed that LRES is present in many cancer types. Here we utilize Illumina Beadchip methylation array analysis to identify LRES across 800 kb of chromosome 5q31 in colorectal adenomas and carcinomas (n=34) relative to normal colonic epithelial DNA (n=6). This region encompasses 53 individual protocadherin (PCDH) genes divided among three gene clusters. Hypermethylation within these gene clusters is asynchronous; while most PCDH hypermethylation occurs early, and is apparent in adenomas, PCDHGC3 promoter methylation occurs later in the adenoma-carcinoma transition. PCDHGC3 was hypermethylated in 17/28 carcinomas (60.7%) according to methylation array analysis. Quantitative real-time reverse transcription-polymerase chain reaction showed that PCDHGC3 is the highest expressed PCDH in normal colonic epithelium, and that there was a strong reciprocal relationship between PCDHGC3 methylation and expression in carcinomas (R=-0.84). PCDH LRES patterns are reflected in colorectal tumour cell lines; adenoma cell lines are not methylated at PCDHGC3 and show abundant expression at the mRNA and protein level, while the expression is suppressed in hypermethylated carcinoma cell lines (R=-0.73). Short-interfering RNA-mediated reduction of PCDHGC3 led to a decrease of apoptosis in RG/C2 adenoma cells, and overexpression of PCDHGC3 in HCT116 cells resulted in the reduction of colony formation, consistent with tumour suppressor capabilities for PCDHGC3. Further functional analysis showed that PCDHGC3 can suppress Wnt and mammalian target of rapamycin signalling in colorectal cancer cell lines. Taken together, our data suggest that the PCDH LRES is an important tumour suppressor locus in colorectal cancer, and that PCDHGC3 may be a strong marker and driver for the adenoma-carcinoma transition.

Decoding key nodes in the metabolism of cancer cells: sugar & spice and all things nice

In the past 5 years, a convergence of studies has resulted in a broad appreciation in the cancer research community that reprogramming of cellular metabolism may be more central to cancer than appreciated in the past 30 years. The re-emergence of cancer metabolism stems in part from discoveries that a number of common oncogenes and tumor suppressor genes more directly control cell metabolism than previously thought. In addition, a number of what would previously have been called “card-carrying” metabolic enzymes have been identified as human tumor suppressors or oncogenes, causally mutated in a variety of human cancers. This growing appreciation of the role of altered cell metabolism has led to further investigation into the rate-limiting proteins involved in different aspects of the unique metabolism of tumor cells. Targeting cancer metabolism with drugs requires a therapeutic window in which tumor cells, compared to normal tissues, have a greater dependence on specific metabolic enzymes. Themes that have emerged in the past decade of developing oncogene-targeted cancer therapeutics suggest that tumors with distinct oncogenic lesions are likely to require drugs that target distinct metabolic pathways. Ultimately, the hope is that detailed knowledge of oncogene and tumor suppressor gene functions and their effects on metabolism will lead to drug combinations that will be far more effective in treating cancers.

Coexistent Non-Hodgkin’s Lymphoma and Renal Cell Carcinoma in a Patient with Von Hipple-Lindau Disease: A Case Report

Von Hippel-Lindau (VHL) disease is an autosomal dominant syndrome that, due to loss of tumor suppressor gene function, predisposes affected individuals to various benign and malignant tumors including renal cell cancer. In contrast, lymphomas are a varied group of clonal diseases arising from a lymphocyte progenitor and can affect any site of the lymphatic system. We present the case of a 56 year old female with Von Hippel-Lindau disease and clear cell renal carcinoma (CCRC), who developed a nasopharyngeal mass with cervical and submandibular lymphadenopathy subsequently proven to be a Non Hodgkin’s lymphoma (NHL). Though the association between VHL and renal cell cancer is significant, a mechanism linking these two diseases has previously been unknown. To the best of our knowledge, this is the first reported case of a patient presenting with coexistent VHL and NHL. In an effort to explain this rarity, we propose that a defective VHL allele may serve as a possible link between VHL and RCC, thus leading to an environment that would favour the development of a malignant clone, our patient’s NHL.

Prognostic Significance of Regulators of Cell Cycle and Apoptosis, p16INK4a, p53, and bcl-2 in Primary Mucosal Melanomas of the Head and Neck

Abnormalities in cell cycle regulation, tumor suppressor gene functions and apoptosis are frequent events in tumorigenesis. Their role in the pathogenesis and prognosis of primary mucosal melanomas (MM) of the upper aerodigestive tract remains unknown. Sixty-four patients (40 men, 24 women, median age 64 years) with MM were included in this study; 32 had tumors in the nasal/paranasal cavities, 28 in the oral cavity and 4 in the pharynx. Archival tissues from 47 initial mucosal tumors, 17 mucosal recurrences, and 13 nodal/distant metastases were subjected to immunohistochemistry using antibodies against p16, p53, and bcl-2. The results were correlated with histological features and survival data. Expressions of p16, p53, and bcl-2 proteins were seen in 25% (N=19/76), 21% (N=16/76), and 74% (N=56/76) of all tumors, respectively. bcl-2 expression in the initial tumors was associated with significantly longer overall and disease specific survival (3.3 vs. 1.5 years, P ≤ 0.05). Expression of p16 was increasingly lost, from 32% in initial tumors to 12% in recurrent and 15% in metastatic tumors (P=0.06). Tumors comprised of undifferentiated cells were significantly more p53 positive than epithelioid or spindle cells (80% vs. 33%, P=0.02). Expression of these markers did not correlate with necrosis, or vascular and/or deep tissue invasion. Expression of bcl-2 is associated with better survival in MM. Loss of p16 was seen with tumor progression whereas aberrant p53 expression was frequent in undifferentiated tumor cells.

Complex Regulation of p73 Isoforms after Alteration of Amyloid Precursor Polypeptide (APP) Function and DNA Damage in Neurons

Genetic ablations of p73 have shown its implication in the development of the nervous system. However, the relative contribution of ΔNp73 and TAp73 isoforms in neuronal functions is still unclear. In this study, we have analyzed the expression of these isoforms during neuronal death induced by alteration of the amyloid-β precursor protein function or cisplatin. We observed a concomitant up-regulation of a TAp73 isoform and a down-regulation of a ΔNp73 isoform. The shift in favor of the pro-apoptotic isoform correlated with an induction of the p53/p73 target genes such as Noxa. At a functional level, we showed that TAp73 induced neuronal death and that ΔNp73 has a neuroprotective role toward amyloid-β precursor protein alteration or cisplatin. We investigated the mechanisms of p73 expression and found that the TAp73 expression was regulated at the promoter level. In contrast, regulation of ΔNp73 protein levels was regulated by phosphorylation at residue 86 and multiple proteases. Thus, this study indicates that tight transcriptional and post-translational mechanisms regulate the p73 isoform ratios that play an important role in neuronal survival.

Differences between oral cancer and cancers of the pharynx and larynx on a molecular level

In order to elucidate differences between oral cancers and cancers of the pharynx and larynx, we investigated the genetic and epigenetic changes in these tumors using molecular biology methods. Methylation of the promoter region of the p16 tumor suppressor gene was examined using methylation-specific polymerase chain reaction in specimens from 47 oral, 39 pharyngeal and 35 laryngeal squamous cell carcinomas. These specimens were also characterized for allelic loss of certain areas of the genome, i.e., 3p22, 9p21 and 17p13 (TP53). The frequency of methylation of the promoter region of the p16 gene in tongue cancers (35.3%) was significantly higher than in pharyngeal (12.8%) and laryngeal cancers (11.4%) (p=0.046 and p=0.039, respectively). The frequency of methylation in tumors of female patients (47.1%) was significantly higher compared to tumors of male patients (15.4%) (p=0.0067). In contrast, the frequency of the loss of heterozygosity (LOH) at 3p21 in pharyngeal cancers (66.7%) was significantly higher than in oral cancers (20.0%) (p=0.0006). The frequencies of LOH at 17p13 in pharyngeal (71.0%) and laryngeal cancers (73.1%) were also significantly higher than in oral cancers (36.1%) (p=0.009 and p=0.009, respectively). Our results indicate that there are marked differences in the frequencies of the hypermethylation of genes and allelic loss between oral cancers and cancer of the pharynx and larynx. Although all of these tumors were diagnosed as squamous cell carcinomas, the process of carcinogenesis may be different in tumors located in various parts of the head and neck. Loss of function of tumor suppressor genes by allelic loss gives rise to tumors in the pharynx and larynx, while loss of function due to methylation of the promoter regions of those genes is related to carcinogenesis in the oral cavity.

Molecular insights into the proliferation and progression mechanisms of the oral cancer: Strategies for the effective and personalized therapy

Summary Oral carcinogenesis is a multifactorial process involving numerous genetic events that alter normal functions of oncogenes and tumor suppressor genes. It is well established that an accumulation of genetic alterations is the basis for the progression from a normal cell to a cancer cell, referred to as multi-step carcinogenesis. This event may be increased the production of growth factors or the number of receptors on the cell surface, and/or increased transcription factors or intracellular signal messengers, further that associated with deregulated control of cell proliferation and apoptosis. Together with the loss of tumor suppressor activity, these changes lead to a cell phenotype that can increase cell proliferation, with loss of cell cohesion, and infiltration to adjacent tissue thus causing distant metastasis. Molecular medicine is responsible for defining the molecular mechanisms that underlie the onset of oral cancer. This review focuses on recent advances in our understanding of the molecular control of the innumerable pathways related to these processes. These may lead to short- or medium term improvements in the diagnosis and prognosis of oral cancerous lesions and to the development of novel therapeutic approaches to this disease.

UP-02.186 Identification of MAPK10 as Novel Epigenetic Marker for Chromophobe Kidney Cancer

Chromophobe renal cell carcinoma (RCC) is a distinctive histologic subtype of RCC that appears to be derived from the cortical portion of the collecting duct and has a better prognosis than clear cell RCC. These differences suggest different genetic factors between subtypes of RCC. The epigenetic gene silencing, DNA methylation, leads to a block in the production of functional protein, and makes carcinogenesis to inhibit the function of tumor-suppressor gene. The aim of this study is to detect new hypermethylated genes related with chromophobe RCC of 807 cancer-related genes.

Splice variants of mda-7/IL-24 differentially affect survival and induce apoptosis in U2OS cells

Interleukin-24 (mda-7/IL-24) is a cytokine in the IL-10 family that has received a great deal of attention for its properties as a tumor suppressor and as a potential treatment for cancer. In this study, we have identified and characterized five alternatively spliced isoforms of this gene. Several, but not all of these isoforms induce apoptosis in the osteosarcoma cell line U2OS, while none affect the survival of the non-cancerous NOK cell line. One of these isoforms, lacking three exons and encoding the N-terminal end of the mda-7/IL-24 protein sequence, caused levels of apoptosis that were higher than those caused by the full-length mda-7/IL-24 variant. Additionally, we found that the ratio of isoform expression can be modified by the splice factor SRp55. This regulation suggests that alternative splicing of mda-7/IL-24 is under tight control in the cell, and can be modified under various cellular conditions, such as DNA damage. In addition to providing new insights into the function of an important tumor suppressor gene, these findings may also point toward new avenues for cancer treatment.

Developmentally arrested structures preceding cerebellar tumors in von Hippel–Lindau disease

There is increasing evidence that suggests that knockout of tumor-suppressor gene function causes developmental arrest and protraction of cellular differentiation. In the peripheral nervous system of patients with the tumor-suppressor gene disorder, von Hippel–Lindau disease, we have demonstrated developmentally arrested structural elements composed of hemangioblast progenitor cells. Some developmentally arrested structural elements progress to a frank tumor, hemangioblastoma. However, in von Hippel–Lindau disease, hemangioblastomas are frequently observed in the cerebellum, suggesting an origin in the central nervous system. We performed a structural and topographic analysis of cerebellar tissues obtained from von Hippel–Lindau disease patients to identify and characterize developmentally arrested structural elements in the central nervous system. We examined the entire cerebella of five tumor-free von Hippel–Lindau disease patients and of three non-von Hippel–Lindau disease controls. In all, 9 cerebellar developmentally arrested structural elements were detected and topographically mapped in 385 blocks of von Hippel–Lindau disease cerebella. No developmentally arrested structural elements were seen in 214 blocks from control cerebella. Developmentally arrested structural elements are composed of poorly differentiated cells that express hypoxia-inducible factor (HIF)2α, but not HIF1α or brachyury, and preferentially involve the molecular layer of the dorsum cerebelli. For the first time, we identify and characterize developmentally arrested structural elements in the central nervous system of von Hippel–Lindau patients. We provide evidence that developmentally arrested structural elements in the cerebellum are composed of developmentally arrested hemangioblast progenitor cells in the molecular layer of the dorsum cerebelli.

Homozygously deleted gene DACH1 regulates tumor-initiating activity of glioma cells

Loss or reduction in function of tumor suppressor genes contributes to tumorigenesis. Here, by allelic DNA copy number analysis using single-nucleotide polymorphism genotyping array and mass spectrometry, we report homozygous deletion in glioblastoma multiformes at chromosome 13q21, where DACH1 gene is located. We found decreased cell proliferation of a series of glioma cell lines by forced expression of DACH1. We then generated U87TR-Da glioma cells, where DACH1 expression could be activated by exposure of the cells to doxycycline. Both ex vivo cellular proliferation and in vivo growth of s.c. transplanted tumors in mice are reduced in U87TR-Da cells with DACH1 expression (U87-DACH1-high), compared with DACH1-nonexpressing U87TR-Da cells (U87-DACH1-low). U87-DACH1-low cells form spheroids with CD133 and Nestin expression in serum-free medium but U87-DACH1-high cells do not. Compared with spheroid-forming U87-DACH1-low cells, adherent U87-DACH1-high cells display lower tumorigenicity, indicating DACH1 decreases the number of tumor-initiating cells. Gene expression analysis and chromatin immunoprecipitation assay reveal that fibroblast growth factor 2 (FGF2/bFGF) is transcriptionally repressed by DACH1, especially in cells cultured in serum-free medium. Exogenous bFGF rescues spheroid-forming activity and tumorigenicity of the U87-DACH1-high cells, suggesting that loss of DACH1 increases the number of tumor-initiating cells through transcriptional activation of bFGF. These results illustrate that DACH1 is a distinctive tumor suppressor, which does not only suppress growth of tumor cells but also regulates bFGF-mediated tumor-initiating activity of glioma cells.

Identification of Mirnas and Their Gene Targets Differentially Expressed in Microsatellite Stable and Unstable Colorectal Cancers Through an Integrated Analysis

DE in stably transfected MKN28 cells. Among the 33 differential protein spots, nine were functional importance and further validated by real-time RT-PCR and/or western blot. The tumor suppressive effect of ZNF331 is mediated by down-regulation of genes involved in cell growth (EIF5A, GARS, DDX5, STAM, UMPS) and migration/invasion (ACTR3, DSTN), and up-regulation of genome stability gene (SSBP1) and cellular senescence gene (PNPT1). Conclusion: These results suggest that ZNF331 possesses important functions for the suppression of gastric cancer and can act as a functional tumor suppressor gene in gastric carcinogenesis. Acknowledgment The project was supported by Research Grant ERG CUHK (GRF 473008).