Potential Tumor Suppressor Gene Research Articles

Reprimo as a modulator of cell migration and invasion in the MDA-MB-231 breast cancer cell line.

BackgroundReprimo (RPRM), a highly glycosylated protein, is a new downstream effector of p53-induced cell cycle arrest at the G2/M checkpoint, and a putative tumor suppressor gene frequently silenced via methylation of its promoter region in several malignances. The aim of this study was to characterize the epigenetic inactivation and its biological function in BC cell lines.MethodsThe correlation between RPRM methylation and loss of mRNA expression was assessed in six breast cancer cell lines by methylation specific PCR (MSP), 5′-Aza-2′-deoxycytidine treatment and RT-PCR assays. MDA-MB-231 cells were chosen to investigate the phenotypic effect of RPRM in cell proliferation, cell cycle, cell death, cell migration and invasion.ResultsIn the cancer methylome system (CMS) (web-based system for visualizing and analyzing genome-wide methylation data of human cancers), the CpG island region of RPRM (1.1 kb) was hypermethylated in breast cancer compared to normal breast tissue; more interesting still was that ERα(+) tumors showed higher methylation intensity than ERα(−). Downregulation of RPRM mRNA by methylation was confirmed in MDA-MB-231 and BT-20 cell lines. In addition, overexpression of RPRM in MDA-MB-231 cells resulted in decreased rates of cell migration, wound healing and invasion in vitro. However, RPRM overexpression did not alter cell viability, phosphatidylserine (PS) translocation or G2/M cell cycle transition.ConclusionTaken together, these data suggest that RPRM is involved in decreased cell migration and invasion in vitro, acting as a potential tumor suppressor gene in the MDA-MB-231 cell line.

Comparative Modeling, Molecular Docking, and Revealing of Potential Binding Pockets of RASSF2; a Candidate Cancer Gene.

RASSF2, potential tumor suppressor gene, acts as a KRAS-specific effectors protein and may promote apoptosis and cell cycle arrest. It stabilizes STK3/MST2 by protecting it from proteasomal degradation. RASSF2 plays a significant role against the inhibition of cancer. MODELLER (9v15) and online servers (I-Tasser, SwissModel, 3D-JigSaw, ModWeb) were utilized to generate 3D structures of the RASSF2 based on homology modeling. A comparison between models predicted by MODELLER (9v15) and Web servers had been checked through utilized evaluation tools. The most potent model for RASSF2 was analyzed and selected for molecular docking studies. The binding pockets were revealed for binding studies through Site Hound. AutoDock Vina and AutoDock4 were utilized for molecular docking, and the attempt of this experiment was to identify the ligands for RASSF2. The selected compounds may act as regulators and regulate the normal activity of RASSF2. It was also analyzed and observed that the selected compounds showed least binding energy and high-affinity binding in predicted top binding domain. The determination of protein function is based on accurate identification of binding sites in protein structures. The binding site is known, and it may allow the ligand type and protein function to be determined by performing in silico and experimental procedures. The detection, comparison, and analysis of binding pockets are pivotal to drug discovery. It proposed that predicted structure is reliable for the structural insights and functional studies. The predicted binding pockets may lead to further analysis (drug discovery), used against cancer study.

Protein tyrosine phosphatases receptor type D is a potential tumour suppressor gene inactivated by deoxyribonucleic acid methylation in paediatric acute myeloid leukaemia.

Protein tyrosine phosphatases receptor type D (PTPRD) is a tumour suppressor gene, and its epigenetic silencing is frequently found in glioblastoma. As aberrant deoxyribonucleic acid (DNA) methylation patterning has been shown to play a role in leukaemogenesis, we studied the promoter methylation, expression profiles and molecular functions of PTPRD in paediatric patients with acute myeloid leukaemia (AML). Bone marrow specimens were obtained from 32 Chinese patients with a mean age of 7.2 years (range 1.1-16.5). PTPRD and methylation status were evaluated by real-time polymerase chain reaction (PCR) and methylation-specific PCR. Western blot and flow cytometry techniques were also used. PTPRD expression was decreased by promoter region methylation in six AML cells and methylated in 21 (65.6%) of the 32 samples. In addition, PTPRD expression could be induced by the DNA demethylating agent 5-aza-2'-deoxycytidine. Furthermore, functional studies showed that overexpression of PTPRD in AML cells inhibited cell proliferation and clonogenicity as well as inducing apoptosis. However, PTPRD knockdown increased cell proliferation. These effects were associated with downregulation of cyclin D1, c-myc and upregulation of Bax. The results of this study demonstrated that PTPRD was a potential tumour suppressor gene inactivated by DNA methylation in paediatric AML.

P53 signaling is involved in leptin-induced growth of hepatic and breast cancer cells.

Leptin, an adipokine predominantly produced from adipose tissue, is well known to induce tumor growth. However, underlying molecular mechanisms are not established yet. While p53 has long been well recognized as a potent tumor suppressor gene, accumulating evidence has also indicated its potential role in growth and survival of cancer cells depending on experimental environments. In the present study, we examined if p53 signaling is implicated in leptin-induced growth of cancer cells. Herein, we demonstrated that leptin treatment significantly increased p53 protein expression in both hepatic (HepG2) and breast (MCF-7) cancer cells without significant effect on mRNA expression. Enhanced p53 expression by leptin was mediated via modulation of ubiquitination, in particular ubiquitin specific protease 2 (USP2)-dependent manner. Furthermore, gene silencing of p53 by small interfering RNA (siRNA) suppressed leptin-induced growth of hepatic and breast cancer cells, indicating the role of p53 signaling in tumor growth by leptin. In addition, we also showed that knockdown of p53 restored suppression of caspase-3 activity by leptin through modulating Bax expression and prevented leptin-induced cell cycle progression, implying the involvement of p53 signaling in the regulation of both apoptosis and cell cycle progression in cancer cells treated with leptin. Taken together, the results in the present study demonstrated the potential role of p53 signaling in leptin-induced tumor growth.

Decreased Tumor Suppressor Candidate 3 Predicts Poor Prognosis of Patients with Esophageal Squamous Cell Carcinoma.

TUSC3 was recently identified as a potential tumor suppressor gene in a variety of human malignancies. However, no data are currently available regarding the expressions of TUSC3 in esophageal cancer (ESCC).The purposes of this study was to investigated the expressions of TUSC3 in ESCC tissues and assess the relationship between TUSC3 levels and clinico-pathological characteristics of ESCC patients. TUSC3 protein expressions were evaluated by immunohistochemistry (IHC) on tissue microarray slides in esophageal cancer, which included 95 esophageal squamous carcinoma specimens (ESCC), and 75 normal esophageal mucosa (NEM). We found that TUSC3 in ESCC was significant lower than that in NEM (P=0.000). According to multi-clinical classifications, TUSC3 level varied significantly with TNM stage, T stage, and N stage (p<0.001, p=0.0368, p<0.0001, respectively). Univariate analysis showed that gender, TNM stage, T stage, N stage, TUSC3 expression were prognostic factors for survival. Multivariate analysis showed that in our study, only TUSC3 expression was independent prognostic factors for ESCC. Our results indicated for the first time, a combined analysis of TUSC3 expressions as well as the clinical variables will help predict the prognosis of ESCC patients. Further large-sample validation and functional analysis should be performed to evaluate its potential prognostic and therapeutic values for ESCC patients.

Tumor Suppressor Analysis in CML.

Retroviral models have tremendously contributed to our understanding of CML development and have been indispensable for preclinical drug testing which facilitated the implementation of a targeted therapy. The retroviral insertion of Bcr-Abl into mice that are genetically depleted for a potential tumor suppressor is a tool to test for a specific gene function in Bcr-Abl disease. Here we describe how to generate a Bcr-Abl retrovirus that is subsequently used for infection of primary murine BM cells, which are genetically depleted for a potential tumor suppressor gene. We will suggest control experiments and outline further methods that are required to allow for assessment of disease development upon tumor suppressor knockout in CML.

MiR-96 induces cisplatin chemoresistance in non-small cell lung cancer cells by downregulating SAMD9.

Cisplatin is effective as a single agent or in combination with other drugs for the treatment of non-small cell lung cancer (NSCLC). A concerning clinical challenge with cisplatin-based NSCLC chemotherapy is the intrinsic and acquired chemoresistance to cisplatin. The sterile α motif domain-containing (SAMD9) gene has been reported as a potent tumor suppressor gene that inhibits tumorigenesis and progression of NSCLC. microRNAs (miRNA) have been revealed to play important roles in the regulation of cancer chemoresistance. To the best of our knowledge the present study explored the role of miRNA/SAMD9 signaling in regulating cisplatin chemoresistance in NSCLC for the first time. Out of the several candidate miRNAs predicted to bind the 3′-untranslated region (UTR) of the SAMD9 gene, miRNA-96 (miR-96) demonstrated significant target-sequence-specific inhibition of the SAMD9 3′-UTR luciferase reporter activity in NSCLC cells. In addition, while NSCLC tumor samples exhibited significantly higher expression levels of miR-96 compared with adjacent normal tissues, the expression levels of SAMD9 were significantly lower than those in adjacent normal tissues. miR-96 and SAMD9 were overexpressed and knocked down in the human NSCLC H358 and H23 cell lines and the half maximal inhibitory concentration (IC50) of cisplatin and cell apoptosis rate under cisplatin treatment were used as measures of cisplatin chemoresistance. The present results identified that overexpression of miR-96 in NSCLC cells markedly decreased SAMD9 expression and cisplatin-induced apoptosis, and increased the cisplatin IC50, which could be eliminated by overexpression of SAMD9. By contrast, knocking down miR-96 in NSCLC cells using antagomir-96 significantly increased SAMD9 expression and the cisplatin-induced apoptosis and decreased cisplatin IC50, which could be completely reversed by a knockdown of SAMD9. In conclusion, the current study demonstrates that miR-96 targets and downregulates SAMD9 in NSCLC, which decreases cisplatin-induced apoptosis and induces cisplatin chemoresistance in NSCLC cells. The findings of the present study add novel insights into the function of miR-96 and SAMD9 in cancer, as well as into the molecular mechanisms underlying NSCLC chemoresistance.

Estrogen receptor β expression and colorectal cancer: a systematic review and meta-analysis.

Estrogen receptor β (ERβ) is a potential tumor-suppressor gene in colorectal cancer (CRC). This hypothesis is supported by clinical and laboratory observations. In this meta-analysis, we looked at studies that investigated the relationship between ERβ protein expression and CRC, comparing the lesion with normal adjacent mucosa. English medical literature searches were performed for ERβ expression in patients with CRC, tumor tissue versus normal mucosa. Searches were performed up to 31 May 2015, using MEDLINE, PubMed, EMBASE, Scopus, and CENTRAL. Meta-analysis was carried out using Comprehensive Meta-analysis Software. Pooled odds ratios and 95% confidence intervals were calculated and ERβ expression was compared in individual studies using the fixed-effects model. The odds ratio of ERβ expression was 0.216 (95% confidence interval 0.152-0.307, P<0.0001), lower in cancer tissue than normal mucosa. Funnel plot did not indicate a significant publication bias. There was no significant heterogeneity in the studies included: Q=5.897, d.f.(Q)=9, I=0.000, P=0.750. In this meta-analysis, we confirm the observation of decreased ERβ expression in CRC. Our results support the hypothesis of ERβ being a tumor-suppressor gene in the large bowel, and the ERβ protein protects against carcinogenesis and development of CRC when activated by estrogen. Further studies are needed to examine the potential of selective/specific ligands to activate ERβ without the side effects found with estrogen and without activating ERα. In this meta-analysis, we looked at studies that investigated the relationship between CRC and ERβ expression in the tumor and normal mucosa of CRC patients. English medical literature searches were performed for studies comparing ERβ expression in the cancer and normal colonic mucosa in patients with CRC. Meta-analysis was carried out, pooled odds ratios were calculated, and ERβ expression was compared in individual studies.

Mining for novel tumor suppressor genes using a shortest path approach

Cancer, being among the most serious diseases, causes many deaths every year. Many investigators have devoted themselves to designing effective treatments for this disease. Cancer always involves abnormal cell growth with the potential to invade or spread to other parts of the body. In contrast, tumor suppressor genes (TSGs) act as guardians to prevent a disordered cell cycle and genomic instability in normal cells. Studies on TSGs can assist in the design of effective treatments against cancer. In this study, we propose a computational method to discover potential TSGs. Based on the known TSGs, a number of candidate genes were selected by applying the shortest path approach in a weighted graph that was constructed using protein–protein interaction network. The analysis of selected genes shows that some of them are new TSGs recently reported in the literature, while others may be novel TSGs.

Altered microRNA Expression Profiles and Regulation of INK4A/CDKN2A Tumor Suppressor Genes in Canine Breast Cancer Models.

microRNA (miRNA) expression profiling of cancer versus normal cells may reveal the characteristic regulatory features that can be correlated to altered gene expression in both human and animal models of cancers. In this study, the comprehensive expression profiles of the 277 highly characterized miRNAs from the canine genome were evaluated in spontaneous canine mammary tumor (CMT) models harboring defects in a group of cell cycle regulatory and potent tumor suppressor genes of INK4/CDKN2 family including p16/INK4A, p14ARF, and p15/INK4B. A large number of differentially expressed miRNAs were identified in three CMT cell lines to potentially target oncogenes, tumor suppressor genes and cancer biomarkers. A group of the altered miRNAs were identified by miRNA target prediction tools for regulation of the INK4/CDKN2 family tumor suppressor genes. miRNA-141 was experimentally validated for INK4A 3'-UTR target binding in the CMT cell lines providing an essential mechanism for the post-transcriptional regulation of the INK4A tumor suppressor gene in CMT models. A well-recognized group of miRNAs including miR-21, miR-155, miR-9, miR-34a, miR-143/145, and miR-31 were found to be altered in both CMTs and human breast cancer. These altered miRNAs might serve as potential targets for advancing the development of future therapeutic reagents. These findings further strengthen the validity and use of canine breast cancers as appropriate models for the study of human breast cancers.

PDZRN4 acts as a suppressor of cell proliferation in human liver cancer cell lines.

Recently, some reports show that Ligand of Numb Protein-X 1 (LNX1) could be a suppressor gene in gliomas, while our current research has firstly shown that PDZ domain containing ring finger 4 (PDZRN4), another member of LNX family, could also be a potential suppressor in hepatocellular carcinoma (HCC). PDZRN4, also named LNX4 (Ligand of Numb Protein-X 4), is a member of the LNX family. We recently found that PDZRN4, but not LNX1, was down-regulated in HCC samples, and the role of PDZRN4 in the progression of HCC had not been studied before. To address this question, firstly, we evaluated the expression of PDZRN4 in HCC samples and adjacent non-cancerous tissues. Semi-quantitative polymerase chain reaction showed that PDZRN4 was down-regulated in 24/36 (66.7%) HCC samples separately. In addition, our research shows that PDZRN4 is silenced in all of the 12 HCC cell lines tested. Subsequently, cell-based functional assay exhibited that ectopic expression of PDZRN4 inhibits the proliferation, plate colony formation and anchorage-independent colony formation of HCC cells. Collectively, our results showed that PDZRN4 might be a potential tumour suppressor gene and had anti-proliferative effect on HCC cell proliferation, which would be of great significance to the researches on HCC.

Breast carcinoma metastasis suppressor gene 1 (BRMS1): update on its role as the suppressor of cancer metastases.

BRMS1 was discovered over a decade ago as a potential tumor suppressor gene. In this review, we summarize the recent findings about the structure of BRMS1, mechanisms of its action and a role of BRMS1 in the cancer progression. As a suppressor of metastasis, BRMS1 has demonstrated a variety of ways to act on the cell functions, such as cell migration, invasiveness, angiogenesis, cell survival, cytoskeleton rearrangements, cell adhesion, and immune recognition. This variety of effects is a likely reason behind the robustness of anti-metastatic influence of BRMS1. Intracellular signaling mechanisms employed by BRMS1 include regulation of transcription, EGF/HER2 signaling, and expression of NF-kB, fascin, osteopontin, and IL-6. Recently reported clinical studies confirm that BRMS1 can indeed be used as a prognostic marker. Approaches to employ BRMS1 in a development of anti-cancer treatment have also been made. The studies reviewed here with respect to BRMS1 structure, cellular effects, intracellular signaling, and clinical value consolidate the importance of BRMS1 in the development of metastasis.

Detection of nasopharyngeal carcinoma in Morocco (North Africa) using a multiplex methylation-specific PCR biomarker assay.

BackgroundSilencing of tumor suppressor genes (TSGs) or activation of oncogenes by, e.g., aberrant promoter methylation, may be early events during carcinogenesis. The methylation status of such genes can be used for early detection of cancer. We are pursuing this approach in our efforts to develop markers for early detection and follow-up of nasopharyngeal carcinoma (NPC). We set out to develop this approach to allow identification of NPC from Morocco and then also compared with NPC samples from different geographical locations and different ethnicity with different NPC incidences, Epstein-Barr virus (EBV) prevalence, and environments.ResultsBy multiplex methylation-specific PCR (MMSP), multiple relevant genes can be detected simultaneously, to achieve high sensitivity and specificity. The strong association of EBV with NPC is also very useful in such an approach. We have initially screened for 12 potential marker genes including EBV genes coding for EBV nuclear antigen 1 (EBNA1) and latent membrane protein-1 (LMP1) and ten potential TSGs obtained from previously published data. The resulting assay included EBNA1, LMP1, and three cellular TSGs: ITGA9, RASSF1A, and P16. We evaluated this assay on 64 NPC patient biopsies from Morocco, Italy, and China compared to deoxyribonucleic acid (DNA) from 20 nasopharyngeal control tissues. In the Moroccan NPC cohort (n = 44), prevalence of the EBNA1 gene showed the highest sensitivity (36/44; 82 %) with 94 % specificity. Out of eight (18 %) EBNA1 negative Moroccan samples, only three were positive for at least one methylated cellular gene. By detection of cellular marker genes, the sensitivity increased from 82 to 89 % (39/44). In the whole material of 64 biopsies from three geographical locations, at least any one marker (viral or cellular) could be detected in 91 % of biopsies with 90 % specificity. In a pilot evaluating assay performance on serum DNA from NPC and controls including samples from Italy (n = 11) and China (n = 5), at least any one marker from the MMSP assay could be detected in 88 %, but the specificity was only 50 %.ConclusionsAn MMSP assay has the potential for detection of NPC by screening in high-risk populations. Serum-derived DNA seems not as good as earlier published NPC swab DNA for screening purpose.

Abstract 4943: Engulfment gene GULP1 is a functional tumor suppressor through influencing TGF-β pathway and is silenced by promoter methylation in urothelial carcinoma

Abstract GULP1 functions as a cytoplasmic adaptor protein in the engulfment of apoptotic corpses and cells. It was also reported to be an adapter protein of LRP1 which is known to be a key regulator of TGF-β signaling. TGF- β signaling pathway is known to contribute to urothelial carcinoma (UC) development by increasing invasiveness and metastasis, and inducing epithelial to mescenchymal (EMT) transition. By an integrated genetic approach, we recently identified GULP1 as a potential tumor suppressor gene and epigenetically silenced in ovarian cancer. In the present study we evaluated the promoter methylation of GULP1 in urothelial carcinoma (UC) and functionally characterize GULP1 in urothelial carcinogenesis. We first determined GULP1 protein expression by immunohistochemistry on a customized muscle-invasive UC (MIUC) tissue-microarray. The mechanism of gene silencing was probed using quantitative methylation-specific PCR (QMSP). The association between GULP1 expression and promoter methylation was analyzed by using a subset of primary UC and UC cell lines. Effects of forced overexpression and siRNA-mediated GULP1-knockdown were measured by cell proliferation and colony forming assays. Western blot analysis was performed for several key molecules to determine the effect of GULP1 modulation in the respective pathways. Complete loss of GULP1 expression was observed in 89 out of 104 MIUC (85.6%). In primary tumor tissues and cell lines, promoter methylation of GULP1 were inversely correlated with GULP1 expression and reactivation of GULP1 was observed by 5-aza-dC treatment of UC cell lines. Additionally QMSP assay of GULP1 was performed for 98 primary UC tissues of different cell types and 21 normal urothelial samples. Our initial analysis by using an empiric cut off value revealed GULP1 promoter methylation was significantly associated with UC (P = 0.007, Fisher's exact test). To determine the feasibility of GULP1 QMSP assay for non-invasive detection of UC using urine DNA, we tested urine from 58 UC patients and 46 healthy people. As expected, 33 out of 58(56.9%) UC cases were methylation positive while very low level of methylation were detected in 4 out of 46 urine control samples (8.7%) (P&amp;lt;0.001, Fisher's exact test). Effect of in vitro modulation of GULP1 was determined by MTT assay, BrdU assay and colony formation assay. The results consistently revealed that GULP1 had anti-proliferative activities. In order to detect the potential pathway related to GULP1, RNA from T24 and shGULP1 transfected T24 cells were applied to Cancer pathway finder array (Qiagen). Heme oxygenase 1 (HMOX1) solely showed over 3 fold increase in T24shGULP1 cells. Other associated molecules altered due to GULP1 knock-down include TGF-β RI, p-Smad3C, p15 and LRP1. In conclusion, GULP1 is a candidate tumor suppressor in UC carcinogenesis and it may have a potential for non-invasive UC detection and targeting therapy. Citation Format: Masamichi Hayashi, Leonardo O. Reis, Alexander Baras, Leonel Maldonado, Eliza Guida, Evgeny Izumchenko, Mariana Brait, Trinity Bivalacqua, George J. Netto, Wayne Koch, David Sidransky, Mohammad O. Hoque. Engulfment gene GULP1 is a functional tumor suppressor through influencing TGF-β pathway and is silenced by promoter methylation in urothelial carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4943. doi:10.1158/1538-7445.AM2015-4943

Abstract 2985: Systematic identification of sex-linked molecular alterations and therapeutic strategies in cancer

Abstract Background Patient sex is an important factor in determining cancer incidence and prognosis; however, little is known of the catalog of sex-linked molecular alterations in cancer and how these may impact treatment response. To begin to address these questions, we developed a computational framework to identify sex-linked molecular alterations and to predict therapies to target oncogenic expression patterns identified in male or female tumors. Methods We utilized DNA copy number, DNA methylation, and RNA-Seq data from both normal and tumor tissues across a total of 17 cancer types, profiled as part of The Cancer Genome Atlas (TCGA). For each cancer type and each molecular data type, we performed supervised statistical analyses to identify molecular alterations significantly associated with patient sex. We then used the tumor and normal samples to separately identify RNA expression signatures associated with carcinogenesis in tumors from males and females. Based on these results, we performed sex-stratified pathway analyses and sex-stratified Connectivity Map (C-Map) analyses using lincscloud to identify biological pathways differentially activated in each sex and to prioritize drugs to target the oncogenic signatures in each sex. Results Integrating results from across the 17 cancer types in a meta-analytic framework, we identified 6,469 genes with significantly sex-disparate expression, 3,217 genes with significantly sex-disparate copy number alterations, and 275 genes with significantly sex-disparate methylation (All FDR &amp;lt; 0.05). Of these genes, 13 exhibited significantly (FDR &amp;lt; 0.05) sex-differentiated RNA expression, DNA copy number, and DNA methylation, including: CAV2, a potential tumor suppressor gene; AIFM2, a p53-dependent apoptosis-inducing gene; and DDX43, which induces resistance to MEK inhibitors. Next, utilizing both tumor and normal samples stratified by sex, we identified sex-linked oncogenic pathways, including the Liver X Receptor (LXR) and Retinoid X Receptor (RXR) activation pathways, both potential therapeutic targets, which were significantly associated with male but not female neoplastic signatures in hepatocellular carcinoma. Lastly, we constructed sex-specific sensitivity and resistance signatures from RNA-Seq data and used the lincscloud CMAP resource to calculate sex-specific connectivity scores for thousands of drugs and small molecule compounds, leading to the identification of known sex-linked drug sensitivity associations (e.g. Tamoxifen in lung cancer), as well as the prediction of several novel sex-linked drug sensitivity associations. Conclusion In summary, we developed an integrative computational approach for large-scale discovery of sex-differentiated molecular alterations and for rational prediction of therapeutic strategies to target sex-linked oncogenic signatures. Citation Format: Jonathan Ma, Sadhika Malladi, Andrew H. Beck. Systematic identification of sex-linked molecular alterations and therapeutic strategies in cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2985. doi:10.1158/1538-7445.AM2015-2985

Abstract 3929: Overexpression of ZNF703 may activate Akt1 in NSCLC

Abstract Like most other epithelial malignancies, NSCLC is characterized by a complex karyotypic pattern with multiple, non-randomly gains and deletions of chromosomal segments. Therefore, several investigators have analyzed NSCLC tumors using conventional comperative genomic hybridization (CGH) or fluorescence in situ hybridization (FISH) techniques to identify the chromosomal aberrations. The 8p11-12 region is one of the most frequently rearranged chromosomal regions in the human genome. The region is prone to chromosomal breaks which makes it a candidate region for cancer research. It can contribute to oncogenesis via inactivation of one or multiple potential tumor suppressor genes and/or amplification and overexpression of candidate oncogenes. Four different amplicons have been identified in the 8p11-12 region. The ZNF703, PROSC, BRF2 and RAB11F1P1 genes reside on the telomeric amplicon A1 and are considered as potential drivers of this amplicon. ZNF703 is one of the candidate oncogenes and its amplification and/or overexpression has been associated with luminal type breast cancer. ZNF703 belongs to the NET family zinc-finger transcription factors and its function depends on the cellular context. More recently, it has been shown that ZNF703 overexpression can activate the Akt/mTOR signaling pathway in breast cancer cell lines. The Akt1 protein can be activated by different pathways and phosphorylation at theSer473 residue is an indicator of activation. In this study we analyzed the copy numbers and expression levels in association with Akt1 phosphorylation levels by MLPA, qRT-PCR and Western blotting, respectively. ZNF703 amplification was detected in 46.3% of the tumor tissue samples compared to adjacent non-cancerous tissues. When we analyzed the mRNA expression levels of the ZNF703 gene in the tumor samples we observed overexpression in 28 (59.6%) of the samples. We also investigated phosphorylation of the Akt protein in 43 tumor samples in association with ZNF703 expression. An increase in Akt phosphorylation was observed in 51.1% of the tumor tissue samples. When we correlated Akt1 phosphorylation with CNV and ZNF703 mRNA expression levels, the increase of p-Akt1 was more frequent in the samples displaying ZNF703 gene amplification and overexpression. However, this positive association was not statistically significant (p = 0.07). On the other hand, when association of ZNF703 expression and Akt1 phoshorylation levels with clinicopathological parameters were analyzed, higher ZNF703 mRNA levels were observed in SCC tumors compared to adenocarcinomas (p = 0.05). Our results indicate that ZNF703 may contribute to the development of SSCs by activating the Akt signaling pathway. Citation Format: NUR BUYRU, Burak Bakir, Onur Baykara, Kamil Kaynak, Nejat Dalay. Overexpression of ZNF703 may activate Akt1 in NSCLC. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3929. doi:10.1158/1538-7445.AM2015-3929

A compendium of DIS3 mutations and associated transcriptional signatures in plasma cell dyscrasias.

DIS3 is a catalytic subunit of the human exosome complex, containing exonucleolytic (RNB) and endonucleolytic (PIN) domains, recently found mutated in multiple myeloma (MM), a clinically and genetically heterogeneous form of plasma cell (PC) dyscrasia. We analyzed by next-generation sequencing (NGS) the DIS3 PIN and RNB domains in purified bone marrow PCs from 164 representative patients, including 130 cases with MM, 24 with primary PC leukemia and 10 with secondary PC leukemia. DIS3 mutations were found respectively in 18.5%, 25% and 30% of cases. Identified variants were predominantly missense mutations localized in the RNB domain, and were often detected at low allele frequency. DIS3 mutations were preferentially carried by IGH-translocated/nonhyperdiploid patients. Sequential analysis at diagnosis and relapse in a subset of cases highlighted some instances of increasing DIS3 mutation burden during disease progression. NGS also revealed that the majority of DIS3 variants in mutated cases were comparably detectable at transcriptional level. Furthermore, gene expression profiling analysis in DIS3-mutated patients identified a transcriptional signature suggestive for impaired RNA exosome function. In conclusion, these data further support the pathological relevance of DIS3 mutations in plasma cell dyscrasias and suggest that DIS3 may represent a potential tumor suppressor gene in such disorders.

Analysis of an independent tumor suppressor locus telomeric to Tp53 suggested Inpp5k and Myo1c as novel tumor suppressor gene candidates in this region.

BackgroundSeveral reports indicate a commonly deleted chromosomal region independent from, and distal to the TP53 locus in a variety of human tumors. In a previous study, we reported a similar finding in a rat tumor model for endometrial carcinoma (EC) and through developing a deletion map, narrowed the candidate region to 700 kb, harboring 19 genes. In the present work real-time qPCR analysis, Western blot, semi-quantitative qPCR, sequencing, promoter methylation analysis, and epigenetic gene expression restoration analyses (5-aza-2´-deoxycytidine and/or trichostatin A treatments) were used to analyze the 19 genes located within the candidate region in a panel of experimental tumors compared to control samples.ResultsReal-time qPCR analysis suggested Hic1 (hypermethylated in cancer 1), Inpp5k (inositol polyphosphate-5-phosphatase K; a.k.a. Skip, skeletal muscle and kidney enriched inositol phosphatase) and Myo1c (myosin 1c) as the best targets for the observed deletions. No mutation in coding sequences of these genes was detected, hence the observed low expression levels suggest a haploinsufficient mode of function for these potential tumor suppressor genes. Both Inpp5k and Myo1c were down regulated at mRNA and/or protein levels, which could be rescued in gene expression restoration assays. This could not be shown for Hic1.ConclusionInnp5k and Myo1c were identified as the best targets for the deletions in the region. INPP5K and MYO1C are located adjacent to each other within the reported independent region of tumor suppressor activity located at chromosome arm 17p distal to TP53 in human tumors. There is no earlier report on the potential tumor suppressor activity of INPP5K and MYO1C, however, overlapping roles in phosphoinositide (PI) 3-kinase/Akt signaling, known to be vital for the cell growth and survival, are reported for both. Moreover, there are reports on tumor suppressor activity of other members of the gene families that INPP5K and MYO1C belong to. Functional significance of these two candidate tumor suppressor genes in cancerogenesis pathways remains to be investigated.Electronic supplementary materialThe online version of this article (doi:10.1186/s12863-015-0238-4) contains supplementary material, which is available to authorized users.

Unique microRNAs in lung adenocarcinoma groups according to major TKI sensitive EGFR mutation status.

BackgroundLung cancer is the leading cause of cancer mortality, despite development of therapeutic strategies. Altered expression of microRNAs(miRNAs) in human malignancies have been well recognized as diagnostic and prognostic indicators, including lung cancer. This study aims to delineate the clinicopathologic significance of three unique miRNAs in adenocarcinoma according to major sensitive EGFR mutation status.MethodsOne-hundred and three formalin-fixed paraffin-embedded (FFPE) tissues were collected from lung adenocarcinoma patients who underwent surgery and epidermal growth factor receptor (EGFR) mutation study. The samples were divided into three groups which include EGFR mutation in exons 19 and 21 and wild type. Some representative cases from each group were profiled using commercial miRNA microarray plates. Three significant miRNAs were selected and they were validated by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), using collective cases of FFPE samples.ResultsWe identified three microRNAs (miR-34c, miR-183, and miR-210) which showed significantly altered expression in all groups of lung adenocarcinoma by microarray study. Compared to normal control lung tissue, down-regulation of miR-34c and up-regulation of miR-183 and miR-210 were identified in caner groups (p < 0.05 for each). We validated the expression of three miRNAs by qRT-PCR. Expression levels of miR-34c, miR-183, and miR-210 were significantly different between normal control group and cancer groups (p = 0.034, <0.000, and 0.036, respectively). Moreover, expression level of miR-183 was significantly higher in EGFR mutation groups than wild type group (p = 0.028). Higher expression levels of three miRNAs were positively related to poor tumor differentiation. Increased expression of miR-183 was positively associated with lymphovascular invasion (p = 0.037). Aberrant expression of miR-210 was independently associated with T stage (p = 0.019), and TNM stage (p = 0.007). However, there was noted a limited statistical significance. In EGFR exon 19 mutation group, miR-34c high expression group showed poor overall survival than low expression one by univariate Kaplan-Meier method. (p = 0.035).ConclusionsHere, we show that miR-34c may act as a potential tumor suppressor gene and miR-183 and miR-210 have a potential oncogenic role in pulmonary adenocarcinoma. This study also suggests different miRNA expression between EGFR mutation group and wild type group. Consequently, further studies of the biology of miRNAs may lead to diagnostic and prognostic biomarkers in pulmonary adenocarcinoma.

A preliminary study of the effect of ECRG4 overexpression on the proliferation and apoptosis of human laryngeal cancer cells and the underlying mechanisms.

Human esophageal cancer-related gene 4 (ECRG4) is a potential tumor suppressor gene isolated from human esophageal epithelial cells. Studies have shown that ECRG4 effectively inhibits the proliferation of tumor cells and induces apoptosis. However, the role of ECRG4 in laryngeal cancer has not yet been clearly defined. In this study, a human laryngeal cancer cell line stably overexpressing ECRG4 was established. The effect of ECRG4 on the proliferation and apoptosis of laryngeal cancer cells and the associated mechanisms were investigated. The Hep-2 human laryngeal carcinoma cell line exhibited a low basal level of ECRG4 expression and was selected for the present study. The eukaryotic expression plasmid pcDNA3.1-ECRG4 was constructed and introduced into Hep-2 cells by transfection reagents. Western blot analysis, reverse transcription-quantitative polymerase chain reaction and immunofluorescence staining confirmed high-level expression of ECRG4. The 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and colony formation assay showed that ECRG4 over-expression suppressed the proliferative capacity of laryngeal cancer cells in vitro. Cell cycle analysis showed that ECRG4 induced cell cycle arrest at the G0/G1 phase. Flow cytometric analysis and Hoechst staining demonstrated that overexpres-sion of ECRG4 significantly induced apoptosis. Western blot analysis confirmed that Bcl-2-associated X protein, cleaved-caspase-3 and cleaved-poly (ADP-ribose) polymerase were upregulated in the apoptotic process, whereas B-cell lymphoma 2 was downregulated. In conclusion, overexpression of ECRG4 inhibited laryngeal cancer cell proliferation and induced cancer cell apoptosis. Therefore, ECRG4 exhibits potential as an effective target in gene therapy for laryngeal cancer.