Expression Of Suppressor Genes Research Articles

Abstract 1037: Targeting the RBM38-EIF4E complex to increase wild-type p53 as a novel cancer therapeutic strategy

Abstract To date, dysregulation of more than 450 RNA-binding proteins (RBPs) across a multitude of cancers has been implicated in tumorigenesis and chemoresistance. In fact, emerging evidence suggests RBPs are principal modulators of every hallmark of cancer progression. Functionally, RBPs are believed to modulate the stability, localization, and translation of transcripts encoding for virtually all known oncogenes and tumor suppressor genes. Consequently, aberrant expression of RBPs may contribute to the malignant transformation of cancer cells through enhancing the expression of oncogenes or decreasing the expression of tumors suppressor genes, such as TP53. As a potent transcription factor, TP53 is a leading regulator of numerous signaling pathways involved in tumor suppression. While mutation or deletion of TP53 contributes to tumorigenesis in more than half of all human cancers, suppression of wild-type TP53 expression also drives tumorigenesis. Thus far, multiple RBPs have been implicated in the repression of wild-type TP53, such as RNA-binding motif protein 38 (RBM38). Specifically, RBM38 interacts with eukaryotic translation initiation factor 4E (EIF4E) on the TP53 transcript, preventing EIF4E from binding to TP53 m7G cap, thus inhibiting TP53 translation. We previously demonstrated that Pep8, an 8 amino acid peptide derived from RBM38, disrupts the RBM38-EIF4E complex enhancing TP53 expression. Moreover, Pep8 alone or in combination with doxorubicin, significantly decreased tumor xenograft growth. As activation of wild-type TP53 by targeting non-genetic mechanisms that inhibit TP53 expression is a promising therapeutic tactic for malignancies that carry wild-type TP53, we are developing more efficacious Pep8 derivatives as a novel therapeutic approach for cancers that express wild-type TP53. Citation Format: Chris August Lucchesi, Jin Zhang, Xinbin Chen. Targeting the RBM38-EIF4E complex to increase wild-type p53 as a novel cancer therapeutic strategy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1037.

Abstract 13223: Split-Intein Mediated Adeno Associated Virus Delivery of CRISPR/dHFCas9-TET3CD as Antifibrotic Therapy

Introduction: Cardiac fibrosis is characterized by excessive deposition of extracellular matrix for which no specific therapy is available as of yet. Gene methylation plays an important role in the progression of cardiac fibrosis. We previously demonstrated that high-fidelity CRISPR/Cas9-based gene-specific hydroxymethylation rescues gene expression of fibrosis suppressor genes and attenuates renal fibrosis. Its efficacy in treating cardiac fibrosis, however, has not been examined. Hypothesis: While AAV-mediated delivery of CRISPR/Cas9 holds great therapeutic potential to treat cardiac fibrosis, several challenges still need to be overcome. We here aimed to circumvent the limiting viral packaging capacity of AAVs, and to identify the best AAV serotype to target mouse cardiac fibroblasts. Methods: We utilized split-intein mediated protein-splicing to divide dHFCas9-TET3CD (dHFCas9 fused to the catalytic domain of TET3) into two parts in order to perform AAV-mediated gene-specific demethylation of a fibrosis suppressor gene. Moreover, we explored the efficacy of 9 different serotypes in targeting mouse cardiac fibroblasts. To explore the antifibrotic efficacy of the fused dHFCas9-TET3CD in vivo, the Angiotensin-II induced cardiac fibrosis mouse model was used. Results: We identified that AAV9-SLRSPPS has a tropism for mouse cardiac fibroblasts with an efficiency of 72% in vitro and 51% and 32% in vivo after 7 days and 4 weeks, respectively. We showed that upon AAV transduction, the fused dHFCas9-TET3CD protein leads to gene-specific demethylation and attenuates cardiac fibrosis in the Angiotensin-II induced cardiac fibrosis mouse model . Conclusions: We demonstrate the use of a Cas9 derivative (dHFCas9-TET3CD) in combination with the split-intein technology and the AAV9-SLRPPS serotype to perform targeted demethylation both in vitro and in vivo which results in amelioration of cardiac fibrosis in vivo. This new technology holds promise not only for cardiac disease but for any disease associated with hypermethylation as well as for other applications where CRISPR/Cas9 in combination with AAV is useful.

Clinical significance of FBXW7 tumor suppressor gene mutations and expression in human colorectal cancer: a systemic review and meta-analysis

BackgroundVarious studies investigating the clinical significance of FBXW7 mutation and/or expression have yielded inconclusive results in colorectal cancer (CRC) patients. Therefore, the present meta-analysis summarizes previous evidence and evaluates the clinical significance, including the prognostic role, of FBXW7 status in CRCs.MethodsThe meta-analysis was conducted by searching the databases of PubMed, China National Knowledge Infrastructure (CNKI), WANFANG data, Web of Science, Embase, and Web of Science. Pooled odds ratios (ORs) and hazard ratios (HRs) and corresponding 95% confidence intervals (CIs) were calculated to assess the relationships between FBXW7 status and clinicopathological features and survival in CRC, respectively.ResultsTen studies involving 4199 patients met the inclusion criteria and included in our meta-analysis. FBXW7 mutation/low expression was obviously correlated with advanced T stage (OR = 0.44, 95% CI: 0.27–0.74, P < 0.01) and lymph node metastasis (OR = 1.88, 95% CI: 1.40–2.53, P < 0.01), but was not associated with other parameters. Further investigation found that FBXW7 mutation/low expression predicted poor OS (HR = 1.25, 95% CI: 1.06–1.47, P < 0.01), but not DFS in CRC (HR = 1.04, 95% CI: 0.60–1.82, P = 0.88). Subgroup analysis found that FBXW7 status was obviously correlated with OS in cohorts recruited after 2009 (HR = 1.32, 95% CI: 1.17–1.50, P < 0.01), from eastern Asia (HR = 1.27, 95% CI: 1.04–1.55, P = 0.02), detected by immunohistochemistry/qRT-PCR (HR = 1.39, 95% CI: 1.22–1.59, P < 0.01), and analysed with multivariate method (HR = 1.47, 95% CI: 1.25–1.74, P < 0.01).ConclusionsThis study indicates that FBXW7 status, expression level especially, is associated with OS but not DFS in CRC. FBXW7 expression level may function as a prognostic biomarker in CRC.

Downregulation of miR-152 contributes to DNMT1-mediated silencing of SOCS3/SHP-1 in non-Hodgkin lymphoma.

Understanding the molecular mechanisms for the development of non-Hodgkin lymphoma (NHL) will improve our ability to cure the patients. qRT-PCR was applied for the examination of the efficiency of shRNA for DNMT1, the expression of suppressor genes, miRNA-152. The MTT analysis, cell cycle analysis, clonal formation, and apoptotic analysis were used to examine the functions of DNMT1 and miR-152 in lymphoma cells. Methylation-specific polymerase chain reaction (MSP) was used to examine the methylation of tumor suppressor genes. The dual luciferase assay and western blot were used to validate if DNMT1 is the target of miR-152. For the in vivo experiments, the lymphoma cells were injected into the nude mice for quantification of the tumor growth after transfection of miR-152 mimics. Knockdown of DNMT1 by shRNA (sh-DNMT1) in OCI-Ly10 and Granta-159 cells significantly upregulated the expression of tumor suppressor genes (SOCS3, BCL2L10, p16, p14, and SHP-1) via decreasing their methylation level. At the cellular level, we found sh-DNMT1 inhibited the proliferation, clonal formation and cell cycle progression and induced the cell apoptosis of lymphoma cells. Furthermore, we found miR-152 can downregulates the expression of DNMT1 via directly targeting the gene. Overexpression of miR-152 also increased the expression of tumor suppressor genes SOCS3 and SHP-1. And miR-152 also can inhibit the cell proliferation and induce the cell apoptosis. Moreover, we found overexpression of miR-152 significantly repressed the tumor growth with decreased DNMT1 expression and increased expression of tumor suppressor genes in vivo. Our study demonstrates that miR-152 can inhibit lymphoma growth via suppressing DNMT1-mediated silencing of SOCS3 and SHP-1. These data demonstrate a new mechanism for the development of NHL and this may provide a new therapeutic target for NHL.

Abstract 4237: Differences in the frequencies of tumor VHL mutation and HIF-2α expression between black and white patients with clear cell renal carcinoma

Abstract Background: Black Americans have a poorer prognosis for clear cell renal cell carcinoma (ccRCC) than white Americans, potentially due in part to differences in tumor biology. In a recent analysis of The Cancer Genome Atlas (TCGA), tumors from black ccRCC patients had a lower rate of mutation in the VHL tumor suppressor gene and lower expression of hypoxia inducible factors (HIF) than tumors from white ccRCC patients. However, as this study was based on a small number of black patients (N=19) and had limited information on patients' medical histories and risk factors profiles, further investigation is needed. Objective: We evaluated differences in the frequencies of somatic VHL gene mutations and HIF-1α and -2α protein expression between tumors from black and white ccRCC patients. Methods: The investigation utilized formalin-fixed tissue and data collected from patients participating in a case-control study conducted in Chicago and Detroit. We sequenced VHL using the Ion Torrent platform for tumors from 69 black and 98 white patients, and measured tumor HIF-1α and -2α protein expression for 88 black and 240 white patients using immunohistochemistry. Results: Black patients' tumors had a lower frequency of VHL mutation than those of white patients (32% vs. 49%; P = 0.03) as well as a lower frequency of above-median HIF-2α expression (33% vs. 56%; P=0.002). HIF-1α expression did not differ by race (P=0.14). These racial differences persisted after multivariable model adjustment for age, sex, hypertension, chronic kidney disease, body mass index, smoking status, stage, grade, and tumor size [VHL mutation: odds ratio (OR) = 0.44, 95% confidence interval (CI) = 0.19, 0.98; HIF-2α expression: OR = 0.33, 95% CI = 0.18, 0.61]. Conclusions: Our observation that VHL mutation and high HIF-2α expression are less frequent in ccRCC tumors of black vs. white patients confirms the earlier TCGA finding. These findings suggest that ccRCC in black patients is a fundamentally different disease than ccRCC in white patients. Citation Format: Catherine L. Callahan, Lee E. Moore, Petra Lenz, Kendra Schwartz, Julie Ruterbusch, Faith Davis, Wong-Ho Chow, W. Marston Linehan, Maria J. Merino, Stephen M. Hewitt, Nathaniel Rothman, Jonathan N. Hofmann, Michael L. Nickerson, Mark P. Purdue. Differences in the frequencies of tumor VHL mutation and HIF-2α expression between black and white patients with clear cell renal 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 4237.

Abstract PR03: Macrophage phenotype drives tumor program via epigenetic machinery carried in secreted microvesicles

Abstract Macrophage phenotypes are reported to regulate tumor progression, angiogenesis, and metastasis in breast cancer by producing soluble factors modulating these programs. Macrophages also communicate via secreted microvesicles (MVs) which are taken-up by neighboring epithelium. MVs contain mRNAs coding the epigenetic regulating machinery, DNA methyltranferases (DNMTs) and histone deacetylases (HDACs), which augment or silence expression via promoter CpG island methylation. Tie2-expressing monocytes (TEMs) is a subset of monocytes reported to augment tumor angiogenesis and metastasis. Recently, we found that increased levels of colony stimulating factor-1 (CSF1) can expand the TEM population in circulation, enabling an influx into breast tumors. Interestingly, we also found that expansion of TEMs by hypoxia was regulated by HIF-1α; and not HIF-2α, but only once they enter the tumor proper. We hypothesized that MVs secreted from M1 and M2 macrophages or TEMs contain epigenetic regulatory machinery which regulate CpG island methylation and gene expression of tumor suppressor genes (TSGs) and genes driving epithelial-to-mesenchymal transition (EMT). We differentiated M1, M2, and TEMs in vitro from CD14+ monocytes isolated from peripheral blood. These cell populations were confirmed using flow cytometry for CD68 and CD80 for M1, CD163 for M2, and CD14/Tie2 for TEMs, as well as M1 (IL-6 and TNFα) and M2 (IL-10 and mannose receptor-1) gene expression profiles. After, we collected MVs using high speed centrifugation techniques characterized by flow cytometry and isolated their nucleic acid content. Using qRT-PCR, we found differential presence of mRNAs for DNMTs and HDACs between M1, M2, and TEM MVs. We cultured these MVs with MCF-10A normal mammary epithelial cells or BEAS-2B normal lung epithelial cells (target cells) for 24 hours and demonstrated MV uptake using Syto RNASelect (RNA) and DiIC16(3) (lipid membrane) and confocal microscopy. After, we isolated RNA and DNA from the target cells and analyzed DNMTs, HDACs, and EMT mRNA expression as well as methyl-specific PCR for CpG island methylation in the promoters of EMT genes. We found that MVs from M1 macrophages increased DNMTs mRNA expression compared to MVs produced from M2 and quiescent macrophages (M0) as well as untreated target cells. To the contrary, HDACs mRNA expression in these target cells cultured with M1-derived MVs was abrogated compared to target cells cultured with MVs from M2 and M0 macrophages and untreated target cells. As a result of the differential MV-carrying DNMTs and HDACs mRNA transferred to the target cells, we found significant differences in CpG island promoter methylation and resultant gene expression in a signature of EMT genes, including TWIST, WNT5A, VIM, FOXC2, KRT19, STAT3, SNAI1 BMP1, TGFb, DSP, AKT1, NUDT13, and ZEB1. The regulation of EMT and tumor suppressor gene promoter methylation and gene expression in MDA-MB-231 human breast cancer cells, as well as the disparate regulation of methylation and gene expression patterns on these target cells as well endothelial cells (HUVEC) by MVs collected from CD14+/Tie2+ TEMs is ongoing. Our current and ongoing work, we establish that M1 and M2 macrophages, and TEMs, secrete MVs containing distinct epigenetic profiles which are taken-up by target cells to regulate promoter methylation and gene expression of TSGs and genes driving EMT. This program of macrophage function may be important in the progression of solid tumors via inhibition of TSGs and activation of a signature of EMT genes in normal epithelial cells as well as to direct the endothelium to support tumor progression. This abstract is also presented as Poster A31. Citation Format: Duaa Dakhlallah, Ivory Patterson, Amy C. Gross, Randall Evans, Tim D. Eubank. Macrophage phenotype drives tumor program via epigenetic machinery carried in secreted microvesicles. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr PR03. doi:10.1158/1538-7445.CHTME14-PR03

Abstract 2303: Decitabine impact on immunohistochemistry scores for tumor suppressor genes FHIT, WWOX, FUS1 and PTEN in human tumor samples

Abstract Purpose: Since tumor suppressor gene (TSG) expression may be lost through promoter hypermethylation, we assessed whether the demethylating agent decitabine could increase TSG expression in tumor tissues of patients receiving decitabine therapeutically. Experimental Design: Patients on a phase I trial received intravenous 1-hour infusions of decitabine 2.5, 5, or 10 mg/m2/day on days 1-5 and 8-12 each 4-week cycle or 15 or 20 mg/m2/day on days 1-5 each cycle, with filgrastim added at higher doses. Tumor biopsies were done pre day 1 and on day 12 of the first cycle. For the putative TSGs FHIT, WWOX, FUS1 and PTEN, immunohistochemistry (IHC) scores (0-300) were calculated by multiplying the % tumor cells staining by the intensity (0-3+) of staining. IHC scores were correlated with methylation of the LINE-1 repetitive element (as a marker of global DNA methylation, determined by pyrosequencing) and with tumor regression. Results: Twenty-five patients had at least one pre- or post-decitabine biopsy evaluable for expression of at least 1 TSG, including 4 patients with breast cancers, 3 kidney, 3 head &amp; neck, 4 melanomas, 3 thymic and 8 others. With negative staining pre-decitabine (score = 0), number of patients converting to positive staining post-decitabine was 1 of 1 for FHIT, 3 of 6 for WWOX, 2 of 3 for FUS1 and 1 of 10 for PTEN. In tumors with low pre-decitabine TSG scores (&amp;lt;150), expression was higher post-treatment in 8 of 8 cases for FHIT (p=0.014, by Wilcoxon signed rank tests for paired comparisons), 7 of 17 for WWOX (p=0.0547), 7 of 12 for FUS1 (p=0.0726), and 1 of 16 for PTEN (p=0.2034). If FHIT, WWOX and FUS1 were considered together, median pre- vs post-decitabine scores were 60 vs 100 (p=0.0002). Overall, TSG scores did not correlate with LINE-1 methylation, but if pre-decitabine scores for FHIT, FUS1 and WWOX were considered together, tumors with IHC scores = 0 for one of these genes (8 observations) had higher pre-decitabine % LINE-1 methylation than did tumors with IHC scores &amp;gt;0 (58 observations) (median 61.6% vs 45.4%, p=0.0481). TSG expression change did not correlate with LINE-1 methylation change, although tumors converting from negative to positive had a median decrease in LINE-1 methylation of 24%, compared to 6% in those not converting (p=0.069). Five of 15 fully evaluable patients had reductions in tumor diameter (by 0.2% to 33.4%). Of these, 3 had simultaneous increases in 3 TSGs (including the 2 patients with the greatest tumor regression) compared to 2 of 10 with tumor growth (p=0.25). Conclusions: In tumors with low TSG expression, decitabine may be associated with increased expression for the TSGs FHIT, WWOX &amp; FUS1, but not PTEN. Further study will be needed to determine if increased TSG expression is associated with reduced DNA methylation and to determine if increased TSG expression may contribute to any decitabine therapeutic effect. Citation Format: David J. Stewart, Maria I. Nunez, Jaroslav Jelinek, David Hong, Sanjay Gupta, C. Marcelo Aldaz, Jean-Pierre Issa, Razelle Kurzrock, Ignacio I. Wistuba. Decitabine impact on immunohistochemistry scores for tumor suppressor genes FHIT, WWOX, FUS1 and PTEN in human tumor samples. [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 2303. doi:10.1158/1538-7445.AM2014-2303

Metallic Nickel Nanoparticles May Exhibit Higher Carcinogenic Potential than Fine Particles in JB6 Cells

While numerous studies have described the pathogenic and carcinogenic effects of nickel compounds, little has been done on the biological effects of metallic nickel. Moreover, the carcinogenetic potential of metallic nickel nanoparticles is unknown. Activator protein-1 (AP-1) and nuclear factor-κB (NF-κB) have been shown to play pivotal roles in tumor initiation, promotion, and progression. Mutation of the p53 tumor suppressor gene is considered to be one of the steps leading to the neoplastic state. The present study examines effects of metallic nickel fine and nanoparticles on tumor promoter or suppressor gene expressions as well as on cell transformation in JB6 cells. Our results demonstrate that metallic nickel nanoparticles caused higher activation of AP-1 and NF-κB, and a greater decrease of p53 transcription activity than fine particles. Western blot indicates that metallic nickel nanoparticles induced a higher level of protein expressions for R-Ras, c-myc, C-Jun, p65, and p50 in a time-dependent manner. In addition, both metallic nickel nano- and fine particles increased anchorage-independent colony formation in JB6 P+ cells in the soft agar assay. These results imply that metallic nickel fine and nanoparticles are both carcinogenetic in vitro in JB6 cells. Moreover, metallic nickel nanoparticles may exhibit higher carcinogenic potential, which suggests that precautionary measures should be taken in the use of nickel nanoparticles or its compounds in nanomedicine.

Abstract B49: Mechanism of cell death induction by dual targeting of HDACs and lysine specific demethylase, LSD1, in leukemias and brain tumors

Abstract Targeting of histone modifications is clinically feasible due to the development of histone deacetylase inhibitors (HDACi), several of which are FDA-approved. In order to identify strategies to augment activity of HDACi, we tested inhibition of lysine specific demethylase-1 (KDM1A/LSD1) in combination with HDACi in malignancies where single agent activity was limited in clinical trials: acute leukemia and glioblastoma, In brain tumor cell lines, patient derived glioblastoma stem cells, and a series of acute leukemia lines, we find high expression of LSD1 protein. Using either knockdown of LSD1, or chemical inhibition via the FDA approved drug, tranylcypromine, we report significant enhancement of HDACi induced cell death. Multiple HDACi were used including vorinostat, panobinostat and entinostat, and cytoxicity was consistently heightened when combined with LSD1 inhibition. Indicative of selectivity of this therapeutic approach, augmentation of HDACi cytotoxicity occurred exclusively in tumor cells, and not in normal counterparts. Caspase activation is a feature of cell death induction by HDAC and LSD1 inhibition and pan-caspase inhibition blocks cell death. Using chemical inhibitors of caspase-8, -9 and -3 and caspase-8 deficient cells, we demonstrate a reliance upon extrinsic pathways of caspase activation by the combination therapy. However, data obtained with caspase-8 deficient cells showed a lack of protection in cells treated with LSD1 inhibitor combined with pan-HDACi (vorinostat and panobinostat), whereas there was significant protection in cells treated with entinostat (a Class I HDACi) and LSD1 inhibitor. This suggests that pan-HDACi may trigger alternate caspases, such as caspase-10, which are blocked by peptide inhibitors but are still active in caspase-8 deficient cells. To gain additional insight into the contribution of LSD1 to cytotoxicity, an apoptosis focused array and RNA-Seq were conducted. The majority of gene expression changes were seen in cells exposed to HDACi alone. However, some of these gene expression changes were further enhanced when LSD1 was also inhibited. Expression of the tumor suppressor genes, p53 and p73, was significantly reduced by HDACi alone, and by the combination of HDACi and LSD1 inhibition. Hrk, a pro-apoptotic Bcl-2 family member, known to be silenced in glioblastoma, was re-expressed by the combination therapy. Time course analyses indicated the loss of p53, p73 and increased Hrk, was an early event, occurring within 6 hours of exposure to HDACi and LSD1 inhibitors, and preceded caspase activation. Upregulation of Hrk transcript was robust and rapid. We observed a ten-fold increase within 3 h of exposure to these agents. Further studies will evaluate the contribution of these early changes to caspase activation, thereby delineating the pathway by which HDACs and LSD1 can be optimally targeted in leukemia and brain tumor model systems. Citation Format: Melissa Singh, Hayley Donnella, Lorimar Ramirez, Joya Chandra. Mechanism of cell death induction by dual targeting of HDACs and lysine specific demethylase, LSD1, in leukemias and brain tumors. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr B49.

Abstract 4637: Developing a first in class of drug to inhibit protein arginine methyltransferase 5 (PRMT5) enzyme dysregulation in glioblastoma multiforme.

Abstract Glioblastoma Multiforme (GBM) is associated with a dismal prognosis despite intensive multimodal therapy, highlighting the need for novel therapeutic approaches. Chromatin remodeling complexes and associated co-repressors such as histone deacetylases (HDAC), DNA methyltransferases (DNMT) and protein arginine methyltransferase 5 (PRMT5) are involved in silencing tumor suppressor and regulatory gene expression and may contribute to glial cell transformation. PRMT5 silences the transcription of key regulatory genes by symmetric di-methylation (S2Me) of arginine (R) residues on histone proteins (H4R3 and H3R8) and works more efficiently when associated with other co-repressor enzymes. We have previously identified PRMT5 over-expression in GBM cell lines, primary GBM tumors, and GBM that spontaneously develop in a pre-clinical mouse model. The degree of PRMT5 over-expression inversely correlated with survival of GBM patients (r=-0.57, p=0.0001) and correlated with proliferation of GBM cell lines (r=0.81, p&amp;lt;0.0001), PRMT5 works concertedly with HDAC2, methyl-CpG binding domain protein 2 (MBD2) and DNMT3a to silence genes with anti-cancer and immune modulatory activities. PRMT5 silending in GBM cell lines leads to growth arrest, apoptosis and loss of HDAC2, DNMT3a and PRMT5 co-repressor complex recruitment at chemokine gene promoters. These findings provide evidence that PRMT5 is a central repressive element required for maintenance of target gene silencing. Thus, we explored methods to inhibit PRMT5 activity as a novel experimental therapeutic strategy for GBM. A rational design of small molecule compounds to inhibit PRMT5 activity led us to construct an in silico model of the human PRMT5 catalytic domain based on available homologous crystal structures. We screened a library of 10,000 compounds and eight small molecules were identified based on binding energy in the PRMT5 catalytic site. Enzyme inhibition assays showed that a lead compound (BLL1) was capable of selectively inhibiting PRMT5 and not PRMT1 or PRMT7 activity (p&amp;lt;0.0001). BLL1 interfered with maintenance of S2Me-H4R3 and S2Me-H3R8 in GBM cell lines by western blot and confocal microscopy. Dose titration experiments with BLL1 showed a dose-dependent response of inhibition of cellular proliferation in GBM cell lines. Combination treatment of GBM cells with subtoxic doses of BLL1, hypomethylating agent 5-azacitidine, and HDAC inhibitor TSA showed synergistic induction of cell death, loss of the epigenetic mark S2Me-H4R3, and de-repression of the immune modulating chemokine CXCL10. Preclinical in vivo studies have shown favorable toxicity and pharmacokinetic profiles for BLL1. We have successfully developed a first in class drug to selectively target dysregulated PRMT5 enzymatic activity in GBM. We are currently developing drugs with improved selectivity and potency. Citation Format: Fengting Yan, Kate Gordon, Kiran Mahasenan, Mark Lustberg, Lapo Alinari, Christian T. Earl, Balveen Kaur, Chenglong Li, Robert A. Baiocchi. Developing a first in class of drug to inhibit protein arginine methyltransferase 5 (PRMT5) enzyme dysregulation in glioblastoma multiforme. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4637. doi:10.1158/1538-7445.AM2013-4637

Abstract A49: Chemoprevention of esophageal cancer using PCA, a metabolite of black raspberry-derived anthocyanins

Abstract Background: We have shown that black raspberries (BRB) as well as their component anthocyanins (AC) inhibit N-nitrosomethylbenzylamine (NMBA)-induced esophageal tumorigenesis in rats. Like many polyphenolic compounds, the bioavailability of BRB anthocyanins is low. The majority of BRB anthocyanins are metabolized by gut bacteria to produce protocatechuic acid (PCA). PCA has been shown to be bioavailable and also to be chemopreventive. Furthermore, we have shown that BRB and their anthocyanins decrease methylation and increase tumor suppressor gene (TSG) expression; e.g., SFRP and WIF1, Wnt pathway antagonists, as well as p16 and PAX6a, in human colon tumors. The current study was designed to determine if PCA is effective in preventing NMBA-induced rat esophageal tumorigenesis by reducing DNA methylation and increasing expression of TSG(s). Materials and Methods: Male Fischer 344 rats aged 3-5 weeks were randomized into five groups and injected s.c. with either the vehicle (20% DMSO/water) or NMBA (0.35 mg/kg b.w.) three times a week for five weeks. During this time all rats were fed control diet (AIN-76A). At the beginning of week 6, all rats were started on their study diets (Group 1: vehicle + AIN-76A, Group 2: NMBA + AIN-76A, Group 3: NMBA + 3.8 umoles AC/g of AIN-76A, Group 4: NMBA+ 6% BRB in AIN-76A, Group 5: NMBA+ 500 ppm PCA in AIN-76A). The concentration of 3.8 umole AC/g of diet for Group 3 was chosen to compare with past studies, 6% BRB were used to match the anthocyanin content of Group 3, and 500 ppm PCA is the estimated amount of anthocyanins converted to PCA from 3.8 umole AC/g diet in the gut. A total of 9 animals per group were harvested at weeks 15 and 25, and 30 rats per group will be harvested at week 35. Esophagi were harvested and esophageal tumors counted and sized. After counting the tumors, one half of the esophagus was used for histological analysis and the other half for DNA/RNA extraction to investigate relevant TSG expression and methylation. Results: The study is still on going. H&amp;E slide scoring revealed significantly reduced hyperplasia in all test diet groups (BRB, AC, PCA) when compared to the NMBA control group at week 15. At week 25, the test diets had no effect on dysplasia frequency, however, all test diets reduced tumor incidence. NMBA treatment led to an increase in dimethyltransferase 3b (DNMT3b) mRNA expression at week 15 and week 25, and 500 ppm PCA decreased DNMT3b expression at both time points. NMBA treatment caused increased methylation and down-regulated mRNA expression of SFRP4 when compared to the vehicle control group at week 25 but not at week 15. None of the test diets influenced DNA methylation or mRNA expression of SFRP4 in NMBA-treated rats at either week 15 or 25. No differences in methylation status of PTX3 (Pentraxin 3), a cytokine released by phagocytic and dendritic cells and shown to be methylated in human esophageal cancer, was observed across all groups at either time point. Conclusion: These results suggest for the first time that PCA may be protective against NMBA-induced esophageal tumorigenesis in rats. PCA significantly reduced tumor incidence, similar to that of BRB and their anthocyanins, by preventing the conversion of dysplastic lesions into papillomas. Preliminary evidence suggests that PCA influences the expression of a key methyltransferase (DNMT3b) in NMBA-treated rat esophagus. Citation Format: Dan Peiffer, Jibran H. Siddiqui, Chieh-Ti Kuo, Yi-Wen Huang, Noah P. Zimmerman, Li-Shu Wang, Gary D. Stoner, Steven G. Carmella, Ben Ransom, Stephen S. Hecht. Chemoprevention of esophageal cancer using PCA, a metabolite of black raspberry-derived anthocyanins. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr A49.

Multi-step process of human breast carcinogenesis: A role for BRCA1, BECN1, CCND1, PTEN and UVRAG

In the female population in Asia, systematic investigation concerning alterations in cancer-related genes in breast carcinoma is rare, and the correlation among oncogene or suppressor gene expression with tumor cell apoptosis, cell cycle regulation and tumor cell autophagy remains to be clarified. In this study, a tissue microarray consisting of 360 individual samples from three different breast tissues was generated. By comparing the expression of the tumor-suppressor genes (BRCA1, BECN1, CCND1, PTEN and UVRAG) in ductal breast cancer and normal breast tissues, respectively, we were able to assign changes in the expression of these mRNAs to specific stages and allocate them to define the roles in the multi‑step process of breast carcinogenesis. Tumor-suppressor genes, such as BRCA1 and BECN1, usually had lower signals in the carcinomatous tissues (10.2 and 6.6%) compared to the normal tissues (31 and 32.6%), while stronger positive dots (positive cells >30%) usually existed in the normal tissues. The patients in the oldest age group had the lowest expression rate. Only BECN1 and CCND1 expression showed a significant association with patient age (p=0.030 and p=0.003). A significant association was observed between BRCA1 and BECN1 expression and tumor size (p=0.028 and p=0.021). BECN1 gene expression was positively correlated with UVRAG and PTEN expression (p=0.006 and p=0.000). CCND1 was negatively correlated with PTEN, BECN1 and BRCA1 expression (p=0.011, p=0.000 and p=0.000). Abnormal expression of BRCA1, BECN1, CCND1, PTEN and UVRAG may play a role in human breast carcinogenesis through dysregulated mRNA expression. Overexpressed CCND1 may shorten the G1 phase of the cell cycle, suppress cell apoptosis and contribute to the formation of invasive ductal carcinoma (IDC).

Abstract B46: Umbilical cord matrix-derived stem cells control tumor growth by their tumor suppressor and promoter gene expression

Abstract Umbilical cord matrix-derived stem cells (UCMSC) have the potential to treat various diseases including cancer. We have shown that naïve human and rat UCMSC significantly attenuate proliferation of multiple cancer cells. However, our previous study showed that the growth attenuation ability of rat UCMSC is stronger than that of human UCMSC. To clarify their different tumoricidal abilities, differential gene expression profiles were studied by microarray analysis using IIIumina HumanRef-8 V2 BeadChip for human and RatRef-12 BeadChip for rat UCMSC. The differential gene expression profile between untreated human UCMSC and those co-cultured with MDA-MB 231 human breast carcinoma cells was compared with that between untreated rat UCMSC and those co-cultured with Mat B III rat mammary gland carcinoma cells. Strict screening criteria used to identify putative genes associated with UCMSC-dependent tumoricidal activity were as follows: gene expression should (1) be over than 1.5 fold different, (2) encode secretory proteins, and (3) be associated with cell growth regulation. Seventeen genes were identified as being associated with either human or rat UCMSC-dependent tumor growth regulation. Among these genes, eight were up-regulated in both human and rat UCMSC (two being known tumor suppressor and six being putative tumor promoter genes). Seven out of seventeen genes were up-regulated in human UCMSC but not in rat UCMSC (three were identified to be tumor suppressor and four were tumor promoter genes). Two out of the seventeen genes, adipose-differentiation related protein (ADRP) and follistatin (FST), which are known tumor suppressor genes, were specifically up-regulated in rat UCMSC, whereas they were down-regulated in human UCMSC when they were co-cultured with carcinoma cells. These results strongly suggest that the balance of the up-regulation of tumor suppressor genes and down-regulation of tumor promoter genes in UCMSC appear to control tumor growth. Since both ADRP and FST are considered to be tumor suppressor genes and were specifically up-regulated in only rat UCMSC, these two genes expression may play central role in strong tumoricidal activity by rat UCMSC. In support of this hypothesis, suppression of ADRP and FST protein by adding a neutralizing antibody (4 µg/ml) in culture medium of rat UCMSC significantly abrogated their ability to attenuate DNA synthesis. Over-expression of ADRP and FST genes by adenoviral vector (100 MOI) in human UCMSC promoted their ability to suppress the DNA synthesis of MDA-MB 231 cells in [3H]-thymidine uptake assay. Interestingly, ADRP expression in human UCMSC stimulated differentiation to adipose type cell morphology (cell enlargement and oil droplet accumulations in cytoplasm). This result suggests that ADRP may stimulate adiponectin production in vivo thereby attenuating tumor growth. Taken together, these results suggest that both ADRP and FST may be key genes that exhibit stronger tumoricidal ability in rat UCMSC than human UCMSC. This work was supported by the Kansas State University (KSU) Terry C. Johnson Center for Basic Cancer Research, KSU College of Veterinary Medicine Dean's Fund, NIH RR017686, RR15563, CA135599, Kansas Bioscience Authority Collaborative Cancer Research grant and by the Intramural Research Program of the NIH, National Institute on Aging. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B46.

Abstract 190: p53 target gene expression, DNA hypomethylation, and clinical response in MDS patients undergoing decitabine therapy

Abstract The myelodysplastic syndromes (MDS) are a heterogeneous group of blood disorders characterized by peripheral cytopenias, a cellular bone marrow with dysplasia and increased intramedullary apoptosis, and are associated with risk of progression to acute myelogenous leukemia (AML). MDS are often fatal due to complications from cytopenias and/or progression to AML. DNA methyltransferase inhibitors (DNMTi), including 5-azacytidine and 5-aza-2’-deoxycytidine (decitabine) have recently assumed a major role in the clinical management of MDS, and are two of only three FDA-approved treatments for these disorders. The mechanistic basis for the activity of DNMTi in MDS is unclear. We recently reported that decitabine treatment induces the expression of p53-inducible ribonucleotide reductase p53R2/RRM2B in tumor cell lines and MDS patient bone marrow (Link et al., Cancer Research 68; 9358-9366, 2008). Importantly, p53R2 induction was independent of DNA hypomethylation at its promoter region, and the level of p53R2 induction correlated with clinical response in a cohort of 13 patients. These data suggest that important pharmacodynamic responses to DNMTi may be associated with DNA damage and/or p53 activation. To better understand the overall nature of the molecular responses occurring in the bone marrow of MDS patients treated with decitabine, in the current study we examined a variety of pharmacodynamic endpoints including: p53 target gene expression, global DNA methylation, tumor antigen gene expression and methylation, and tumor suppressor gene expression. Specifically, we measured the expression of three classical p53 target genes (p21, GADD45-α, NOXA), LINE-1 repetitive element methylation, the expression and methylation of cancer-germline (CG) antigens including MAGE-A1, NY-ESO-1, XAGE-1, and CTCFL/BORIS, and the expression of the tumor suppressor genes (TSGs) p15INK4b and PDLIM4. Our data reveal that decitabine treatment causes robust induction of p53 target genes in MDS patient bone marrow, which coincides with LINE-1 hypomethylation and clinical responses. In contrast, while CG antigen genes showed some evidence for DNA hypomethylation post-therapy, this did not coincide with CG antigen gene induction. Surprisingly, TSGs were also not induced by decitabine therapy in MDS patient bone marrow. Ongoing studies seek to define p53 mutational status in MDS patients and to determine how this relates to p53 target gene induction and/or clinical response. In summary, our data reveal a connection between p53 target gene activation, global DNA hypomethylation, and clinical response in MDS patients undergoing decitabine therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 190.

Abstract 1584: Targeting protein arginine methyltransferase 5 (PRMT5) enzyme over expression in high grade astrocytomas

Abstract High grade astrocytomas are aggressive brain tumors that are associated with a dismal prognosis and are incurable with a median survival of less than 15 months despite intensive multimodal therapy. The linability to effectively target grade III and grade IV (glioblastoma multiforme, GBM) astrocytomas highlights the need for novel therapeutic approaches. Recent studies have shown that epigenetic regulation of chromatin plays a central role in the control of cell growth, differentiation, and survival. Chromatin remodeling enzymes like histone deacetylases, DNA methyltransferases and protein arginine methyltransferase 5 (PRMT5) are involved in silencing tumor suppressor gene (TSG) expression and may contribute towards cellular transformation. PRMT5 silences the transcription of key regulatory genes by symmetric di-methylation (S2Me) of arginine (R) residues on histone proteins (H4R3 and H3R8) and works more efficiently when associated with other co-repressor enzymes. Eight patient-derived GBM cell lines and 45 primary GBM tumors showed abundant expression of PRMT5 protein. Confocal microscopy and immunohistochemical staining showed the PRMT5 signal to localize primarily to the nucleus. Normal brain tissue, normal human astrocytes and low/intermediate grade astrocytomas failed to show PRMT5 expression. The degree of PRMT5 over expression inversely correlated with survival of GBM patients (r=-0.57, p=0.0001) and correlated with proliferation of GBM cell lines (r=0.81, p&amp;lt;0.0001). Elevated PRMT5 expression was observed in high grade astrocytomas that spontaneously develop in a pre-clinical mouse model of GBM employing conditional Nf1, TP53 and PTEN haploinsufficiency. Small inhibitory RNAs (siRNA) specific for PRMT5 led to loss of PRMT5 protein expression and S2Me of H4R3, a histone target of PRMT5. PRMT5 inhibition led GBM cell lines to cell cycle arrest, apoptosis, and complete loss of cell migratory activity. Apoptosis occurred independent of caspase and p53 pathways. Furthermore, PRMT5 knockdown led GBM cell lines to become sensitized to the toxic effects of temazolomide, a drug frequently used to manage patients with GBM. We utilized gene microarray analysis of cDNA isolated from siRNA (or control RNA) treated GBM cells to identify potential targets of PRMT5 and identified the TSG ST7 and three chemokine transcripts (RANTES, IP10, CXCL11) to be upregulated with PRMT5 knockdown. We used chromatin immuno precipitation to show that siRNA treatment led to loss of PRMT5 recruitment on ST7 and chemokine gene promoters that coincided with restoration of transcriptional and translational activity leading to marked elevation in protein expression. PRMT5 knock-down led to secretion of all chemokines into growth medium. These findings identify PRMT5 as an independent prognostic factor for GBM and an attractive therapeutic target for high grade astrocytomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1584.

Turning Off a MicroRNA Oncogene Cures Lymphoma in Mice

MicroRNAs control the expression of genes. Some microRNAs, called oncomiRs, powerfully affect expression of oncogenes and tumor suppressor genes. MicroRNA-21 (miR-21) is expressed at high levels in many animal and human tumors. A team from Yale University genetically engineered mice to express …

Investigation of Clinical Significance and Expression of Tumor Metastasis-related Genes in The Nasopharyngeal Cancer Using Tissue Microarray Technique*

To investigate the role of abnormal protein expression of the tumor metastasis related-genes in invasion and metastasis of nasopharyngeal cancer(NPC) and to seek the metastasis-related markers for NPC,high-throughput tissue microarray and immunohistochemistry were used to detect protein expression of membrane type 1-matrix metalloproteinase(MT1-MMP),membrane type 2-matrix metalloproteinase(MT2-MMP),membrane-cytoskeletal crosslinker Ezrin(Ezrin),metastasis suppressor gene nm23-H1,the epithelial cell adhesion molecule E-cadherin(E-cad)and tissue inhibitor of metalloproteinase-2(TIMP-2) in NPC.Results showed that significant high expression of MT1-MMP and Ezrin proteins and very low expression of nm23-H1 and TIMP-2 proteins were found in NPC compared with non-cancerous nasopharyngeal epithelium(P 0.05,P 0.01,respectively).Expression of MT1-MMP,MT2-MMP and Ezrin proteins in the stageⅡ,stageⅢ and Ⅳ NPC and NPC with lymph node metastasis is significantly higher than that of in the stageⅠ NPC and NPC without lymph node metastasis(P0.05,P0.01 respectively),but expression of nm23-H1 protein in the stageⅡ,stageⅢ and Ⅳ NPC and NPC with lymph node metastasis is significantly lower than that of in the stageⅠ NPC and NPC without lymph node metastasis(P 0.05).There were significantly positive correlation between MT1-MMP and MT2-MMP(r = 0.308,P 0.001),nm23-H1 and E-cad(r = 0.167,P 0.05),nm23-H1 and TIMP-2(r = 0.279,P = 0.001);E-cad and TIMP-2(r = 0.279,P = 0.001) in the NPC.Also,there were obviously negative correlation between MT1-MMP and E-cad(r=-0.188,P0.05),MT1-MM and TIMP-2(r=-0.233,P0.05),Ezrin and E-cad(r =-0.204,P 0.05) in NPC.Based on the cluster analysis,there was common higher expression of MT1-MMP,MT2-MMP and Ezrin proteins in the NPC than that of in the chronic inflammatory nasopharyngeal epithelium(P0.05).However,common higher loss expression of nm23-H1,E-cad and TIMP-2 proteins were found in NPC compared with the peri-cancer nasopharyngeal epithelium and chronic inflammatory nasopharyngeal epithelium(P 0.05,P 0.01).Logistic regression analysis and validity estimation indicated that MT1-MMP gene might be better independent prognostic value for metastasis and clinical progress of NPC.The results suggested that high protein expression of multiple metastasis related-genes,low expression of the metastasis suppressor genes and loss balance between metastasis genes and metastasis suppressor genes might play important role in the metastasis and clinical progression of NPC.MT1-MMP protein can be used as the better independent prognostic molecular marker for metastasis of NPC.

Tumor suppressor genes expression in breast cancer and their recent research progress

In recent years, the incidence of breast cancer is on the rise, particularly in the economical-ly developed areas. The incidence and development of tumor are associated with the abnormal expression of a number of oncogenes and tumor suppressor genes. Suming up the mutation and expression of breast cancer relat-ed suppressor genes can better understand the pathogenesis of breast cancer. Key words: Breast neoplasms; Genes, tumor suppressor; Mutation

M2035 Ductal Adenocarcinoma of the Pancreas: Expression of Growth Factor Receptors, Oncogenes and Suppressor Genes and Its Relationship to Pathological Features, Staging and Survival

M2035 Ductal Adenocarcinoma of the Pancreas: Expression of Growth Factor Receptors, Oncogenes and Suppressor Genes and Its Relationship to Pathological Features, Staging and Survival

Autophagy: For Better or for Worse, in Good Times or in Bad Times …

Autophagy is a bulk cytosolic degradative process which in the last few years has become a key pathway for the advancement of molecular medicine. Autophagy (cellular self-eating) has several implications in human disorders involving accumulation of cytosolic protein aggregates such as Alzheimer, Parkinson, Huntington diseases, as well as in myopathies caused by deficient lysosomal functions and in cancer. Moreover, autophagy affects intracellular microorganism lifespan, acting either as a cellular defense mechanism or, on the contrary, promoting pathogen replication. Furthermore, autophagy also participates in antigen presentation, as a part of the adaptive immune response. Therefore, autophagy association with cell survival or cell death would depend on cell nutrition conditions, presence of cell intruders, and alterations in oncogene or suppressor gene expression. In this review we will focus on the wide spectra of disease-related topics where autophagy is involved, particularly, in those processes concerning microorganism infections.