bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2081: The CCCTC-binding factor (CTCF) functions to downregulate the transcription of MGMT gene in human glioblastoma cells
Authors: Srivenugopal Kalkunte S. , Al-Obaide Mohammed Ibrahim
Published: August 2015

The O6-methylguanine-DNA methyltransferase (MGMT) gene located at chromosome 10q26 encodes a DNA repair protein that abrogates the therapeutic effects of alkylating agents by preventing the formation of mutagenic lesions or cytotoxic DNA crosslinks. MGMT is highly expressed in human gliomas and its transcriptional regulation has emerged as a major translational focal point in neuro-oncology, because the epigenetic silencing of the MGMT gene which occurs in a subset of these malignancies confers improved treatment responses to both chemo and radiation therapies. While the promoter methylation and its contribution to glioma therapy has been much studied, the regulation of MGMT transcription per se, proteins accessory to the process and the chromatin structure thereof are not well understood. The CCCTC-binding transcription factor (CTCF) is one such multifunctional protein involved not only in DNA methylation but also with transcriptional activation/repression, and induction of short or long range chromatin looping. CTCF, an 11 zinc-finger containing protein is more frequently known for its insulating effects by binding diverse nucleotide sequences and restricting the function of transcriptional enhancers. Our bioinformatic analysis of the human MGMT gene showed an unusually high (7-10 sites) congregation of CTCF-binding sites distal to the promoter in human cancer cell lines. Given these considerations, we used CTCF siRNA to eliminate the transcription factor in two MGMT-proficient GBM cell lines (SF180 and GBM10). A specific reduction of CTCF protein by individual and pooled siRNAs was consistently accompanied by increased MGMT protein and mRNA levels. Furthermore, the DNA repair activity of MGMT was increased by 50%, which correlated with a proportional reduction of the CTCF protein in siRNA-exposed cells. Currently, we are investigating the effect of CTCF on MGMT reactivation in cells with methylation, MGMT promoter activity in CTCF silenced cells and the effect of enforced CTCF expression on MGMT transcription. Our results for the first time highlight the involvement of multivalent CTCF in MGMT expression. Since CTCF is tightly linked with DNA methylation, further studies should reveal its role in silencing of MGMT in human GBMs (supported by CPRIT RP130266 and NIH RO3CA125872 grants to KSS).

Citation Format: Kalkunte S. Srivenugopal, Mohammed Ibrahim Al-Obaide. The CCCTC-binding factor (CTCF) functions to downregulate the transcription of MGMT gene in human glioblastoma cells. [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 2081. doi:10.1158/1538-7445.AM2015-2081
Abstract 2082: Control and function of the PROX1 transcription factor in malignant glioma
Authors: Goudarzi Kaveh M. , Elsir Tamador , Nistér Monica , Lindström Mikael S.
Published: August 2015

Malignant gliomas remain to be fatal and highly resistant to therapy. PROX1 is a transcription factor critical for embryonic development, with a role in cell cycle control and progenitor cell differentiation. PROX1 is highly relevant to study in relation to brain tumors due to its role in normal CNS development, where it leads to progenitor cell differentiation and initiation of neurogenesis. We have previously shown that PROX1 is expressed in human gliomas where there is a positive and statistically significant correlation between PROX1 expression and glioma tumor grade. We have also evaluated the prognostic value of PROX1 in a cohort of patients with grade II gliomas and found that a high number of PROX1 positive cells correlated significantly with poor outcome, a clinically important finding, as such predictors do not exist at present. The mechanism behind PROX1 overexpression in high-grade glioma and its functional consequences remain unclear. To this end we set out to study PROX1 effects on the behavior of glioma cells. We found that glioma cell lines in general expressed only low levels of PROX1 protein and mRNA. However, PROX1 was readily detected in the U343MGa Cl2:6 glioma cell line. This cell line also expresses GFAP and SOX2 and has features in common with glioma stem cells. Depletion of PROX1 in U343MGa Cl2:6 cells using siRNA had a marginal positive effect on cell proliferation in a short term assay. Furthermore, ectopic PROX1 impaired the ability of glioma cells to proliferate as indicated by reduced colony formation and decreased BrdU incorporation. We present experimental evidence showing that PROX1 induces p27. By regulating p27, PROX1 may block the activity of Cdk2/Cyclin E complex, and thus inhibit cell cycle progression. The functional consequences of PROX1 expression on the stemness, migration, and invasion phenotypes of glioma cells remain to be further investigated and preliminary results will be presented.

Citation Format: Kaveh M. Goudarzi, Tamador Elsir, Monica Nistér, Mikael S. Lindström. Control and function of the PROX1 transcription factor in malignant glioma. [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 2082. doi:10.1158/1538-7445.AM2015-2082
Abstract 2092: FOXO1/Sprouty2 pathway regulates vascular tumor growth
Authors: Ayyaswamy Sriram , Du Wa , Anderson Christopher , Phung Thuy L.
Published: August 2015

Vascular tumors are endothelial cell neoplasms with a wide spectrum of clinical presentations, ranging from benign infantile hemangiomas in children to low-grade malignant hemangioendotheliomas and highly aggressive angiosarcomas in adults. To date, the molecular basis of vascular tumor pathogenesis is poorly understood and standard therapy for these tumors have limited clinical efficacy.

Forkhead box protein O1 (FOXO1) is a transcription factor with tumor suppressor function and is dysregulated in human cancer. In this study, we showed that FOXO1 suppressed vascular tumor growth, and mechanistically, the inhibitory effects of FOXO1 were mediated by Sprouty2. FOXO1 expression was reduced in a variety of human vascular tumors examined (infantile hemangioma, hemangioendothelioma and angiosarcoma) as compared with normal blood vessels as determined by western blotting and immunohistochemical stains. Knockdown of FOXO1 gene expression with short hairpin RNA resulted in increased vascular tumor cell migration and proliferation in vitro and in vivo. Conversely, over-expression of constitutively active FOXO1 in these cells suppressed cell growth. We observed that FOXO1 interacted with Sprouty2 promoter in situ in chromatin immunoprecipitation assay and increased Sprouty2 gene expression in tumor cells. Similar to FOXO1, Sprouty2 expression was reduced in vascular tumors. Over-expression of Sprouty2 decreased tumor cell growth and migration. Conversely, knockdown of Sprouty2 increased tumor growth in vitro and in vivo. Knockdown of Sprouty2 in cells with over-expression of constitutively active FOXO1 resulted in reduced tumor growth and “rescued” the FOXO1 phenotype, indicating that Sprouty2 is an important mediator of the biological effects of FOXO1. Microarray gene expression profiling of human angiosarcoma cells with Sprouty2 knockdown together with network data integration using bioinformatics analysis and validated by quantitative PCR revealed important Sprouty2-regulated genes that are involved in angiogenesis, apoptosis and growth signal transduction pathways, including the collagen gene family, Notch signaling pathway and the GTPase IMAP family members.

In summary, these findings demonstrate important growth regulatory role of the FOXO1/Sprouty2 pathway in vascular tumors and highlight the potential roles of novel pathways downstream of Sprouty2 in these tumors.

Citation Format: Sriram Ayyaswamy, Wa Du, Christopher Anderson, Thuy L. Phung. FOXO1/Sprouty2 pathway regulates vascular tumor growth. [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 2092. doi:10.1158/1538-7445.AM2015-2092
Abstract 2095: DEPTOR is a direct NOTCH1 target that regulates proliferation, metabolism and glucocorticoid resistance in T cell leukemia
Authors: Hu Yufeng , Su Hexiu , Ye Qi , Wang Zhaojing , Huang Liang , Wang Qian , Liu Shuangyou , Chen Suning , Zhou Jianfeng , Li Peng , Qing Guoliang , Liu Hudan
Published: August 2015

DEP-Domain Containing mTOR-Interacting Protein (DEPTOR) is frequently overexpressed in multiple myelomas and required for cell survival. However, its implications in other hematological malignancies and the precise mechanisms of regulation remain largely undefined. Here, we systematically analyzed DEPTOR expression in human acute lymphoblastic leukemia (ALL), a hematological malignancy accounting for 80% of pediatric leukemia. Whereas substantially inhibited in B-ALLs, unexpectedly, expression of DEPTOR is significantly elevated in a subset of T-ALLs where NOTCH1 is aberrantly activated. Mechanistically, we show DEPTOR expression is dependent on NOTCH signaling in T-ALL cell lines and primary specimens, and demonstrate NOTCH1 directly binds to and activates human DEPTOR promoter. DEPTOR depletion in T-ALL cells abolishes proliferation, attenuates glucose metabolism, enhances apoptosis and sensitizes resistant cells to γ-secretase inhibitor or dexamethasone treatments. Conversely, ectopically expressed DEPTOR promotes proliferation and aerobic glycolysis, and confers dexamethasone resistance. DEPTOR functions through AKT-dependent mechanisms to sustain cell proliferation and drug resistance, while the AKT-independent roles of DEPTOR appear to promote glucose metabolism. We therefore define a novel mechanism that, in addition to the previously reported HES1/PTEN-mediated regulation of AKT, NOTCH1 alternatively activates AKT through inducing the downstream target gene DEPTOR. Our findings thereby highlight DEPTOR as a critical mediator in leukemogenesis and suggest DEPTOR expression may serve as a potential biomarker predicting glucocorticoid resistance and targeting DEPTOR may achieve better therapeutic benefits.

Citation Format: Yufeng Hu, Hexiu Su, Qi Ye, Zhaojing Wang, Liang Huang, Qian Wang, Shuangyou Liu, Suning Chen, Jianfeng Zhou, Peng Li, Guoliang Qing, Hudan Liu. DEPTOR is a direct NOTCH1 target that regulates proliferation, metabolism and glucocorticoid resistance in T cell leukemia. [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 2095. doi:10.1158/1538-7445.AM2015-2095
Abstract 2098: Genome-wide comparison of PU.1 and Spi-B binding sites in a mouse B lymphoma cell line
Authors: Solomon Lauren A. , Li Stephen K.h. , Piskorz Jan , Xu Li S. , DeKoter Rodney P.
Published: August 2015

Background. The E26-transformation-specific (ETS) transcription factor Spi-B is required for the survival of Activated B Cell-type Diffuse Large B Cell Lymphoma cell lines and is recurrently amplified in B cell lymphoma. The ETS transcription factor PU.1 is highly related to Spi-B. Both PU.1 and Spi-B are expressed in B lymphoma cell lines, and have been demonstrated to redundantly activate transcription of genes required for differentiation and function. We hypothesized that Spi-B and PU.1 occupy similar regions of chromatin within the genome of a B lymphoma cell line. Results. To compare binding regions of Spi-B and PU.1, murine WEHI-279 lymphoma cells were infected with retroviral vectors encoding 3XFLAG-tagged PU.1 or Spi-B. Anti-FLAG chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) was performed. Analysis for high-stringency enriched genomic regions demonstrated that PU.1 occupied 4528 regions and Spi-B occupied 3360 regions. 1900 of these regions exhibited at least 100 base pairs of overlap for both factors. Regions bound by Spi-B and PU.1 were frequently located within genes associated with immune response and activation of B cells. Motif-finding revealed that both transcription factors were predominantly located at the ETS core domain (GGAA), however, other unique motifs were identified when examining regions associated with only one of the two factors. Motifs associated with unique PU.1 binding included POU2F2, while unique motifs in the Spi-B regions contained a combined ETS-IRF motif. Conclusions. Our results suggest that complementary biological functions of PU.1 and Spi-B may be explained by their interaction with a similar set of regions in the genome of B lymphoma cells. However, sites uniquely occupied by PU.1 or Spi-B provide insight into their unique functions.

Citation Format: Lauren A. Solomon, Stephen K.h. Li, Jan Piskorz, Li S. Xu, Rodney P. DeKoter. Genome-wide comparison of PU.1 and Spi-B binding sites in a mouse B lymphoma cell line. [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 2098. doi:10.1158/1538-7445.AM2015-2098
Abstract 2099: FoxM1 drives a feed-forward STAT3-activation signaling loop to promote the self-renewal and tumorigenicity of glioblastoma stem cells
Authors: Zhang Sicong , Gong Ai-hua , Wei Ping , Zhou Aidong , Yao Jun , Yuan Ying , Lang Frederick , Rao Ganesh , Huang Suyun
Published: August 2015

The STAT3 signaling pathway is a nodal hub of gliomagenesis, but the mechanisms underlying elevated expression and activation of STAT3 are largely unknown. Here, we showed that FoxM1 was critical for STAT3 gene expression and protein activation. First, the expression of PDGF-A, one of the growth factor upstream of STAT3 directly correlated with the expression of FoxM1 in GBMs. FoxM1 bound to PDGF-A promoter to upregulate PDGF-A expression in GBM stem cells (GSCs) and mouse neural stem cells (NSCs). FoxM1 maintains stemness of GSCs partially via PDGF-A. Second, data from TCGA showed that GBMs expressed high levels of STAT3 as compared with normal brain. STAT3 expression directly correlated with the expression of FoxM1 in GBMs. FoxM1 regulated STAT3 expression by interacting with the β-catenin/TCF4 complex at STAT3 promoter. FoxM1 deficiency decreased both PDGF-A and STAT3 expressions in GSCs and NSCs and inhibited STAT3 activation by PDGF-A, EGF and IL-6, thereby abolished the stemness and tumorigenesis of GSCs. Moreover, constitutively active STAT3 rescued the inhibitory effects of FoxM1 knockdown on the stemness and tumorignecity of GSCs, suggesting FoxM1 regulates the stemness of GSCs by affecting STAT3 Furthermore, overexpression of FoxM1 reprogrammed glioma cells to GSCs-like cells, whereas inhibition of STAT3 diminished FoxM1-induced stemness and tumorigenesis. Collectively, these findings indicated that FoxM1 regulated the expression of both PDGF-A and STAT3 hence drived a feed-forward STAT3-activation signaling loop which was required for maintenance of self-renewal and tumorigenicity of GSCs.

Citation Format: Sicong Zhang, Ai-hua Gong, Ping Wei, Aidong Zhou, Jun Yao, Ying Yuan, Frederick Lang, Ganesh Rao, Suyun Huang. FoxM1 drives a feed-forward STAT3-activation signaling loop to promote the self-renewal and tumorigenicity of glioblastoma stem cells. [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 2099. doi:10.1158/1538-7445.AM2015-2099
Abstract 2101: The transcription factor POU3F2 is expressed in human gliomas and promotes tumorigenesis in vivo
Authors: Rahman Mohummad Aminur , Liess Lina , Lellahi Mohammad S. , Gjerde Christiane H. , Saed Halala S. , Mutlu Ercan , Zhu Huaiyang , Wang Jian , Enger Per Øyvind
Published: August 2015

The neurodevelopmental transcription factor POU3F2 (also called OCT7 / BRN2) is expressed during neurogenesis. Moreover, POU3F2 expression has been reported as a promoter of proliferation and invasion in malignant melanoma of the skin. Since both melanomas and CNS-malignancies arise in organs of neuro-ectodermal origin, we investigated whether human gliomas expressed POU3F2 and the role of POU3F2 in glioma tumorigenesis. We performed immunohistochemistry of 149 grade II-IV gliomas from our tumor bank, and subsequently performed western blots and qPCR of 12 samples from each grade. In addition, we performed flow cytometric analysis of POU3F2 expression in 5 acutely dissociated tumors. Using lentiviral transfection we established glioma cell lines with POU3F2 overexpression or knock down in order the to investigate the effect of POU3F2 on proliferation, migration and differentiation, both in vitro and in vivo. The mechanism, by which POU3F2 regulates glioma progression, was also investigated. Immunohistochemistry showed that POU3F2 was almost uniformly expressed in human gliomas. Both western blot, qPCR and flow cytometry confirmed expression of POU3F2 in human gliomas. In acutely dissociated tumors, the POU3F2 positive cells displayed an increased cell percentage in the S and G2/M phase of the cell cycle. Moreover, POU3F2 overexpression in the glioma cell lines increased growth rates and colony formation. Overexpression of POU3F2 increased the levels of phosphorylated p-44/42 indicating activation of the ERK1/2 signaling pathway. Increased levels of downstream transcriptional targets c-Myc, Elk1 and Stat3 transcription factors in the POU3F2 overexpressing cell lines confirmed activation of the MAPK signaling pathway. Furthermore, the POU3F2 overexpressing cell lines displayed increased expression of the growth factor FGF2. U0126, an inhibitor of MEK1/2, decreased the cell proliferation and decrease levels of phospho-p44/42, phospho-stat3 and FGF2. Moreover, overexpression of the POU3F2 increases the level of other neurodevelopmental transcription factors associated with a pluripotent state. Ongoing studies aim at further elucidating the multiple roles of POU3F2 in brain tumor progression.

Citation Format: Mohummad Aminur Rahman, Lina Liess, Mohammad S. Lellahi, Christiane H. Gjerde, Halala S. Saed, Ercan Mutlu, Huaiyang Zhu, Jian Wang, Per Øyvind Enger. The transcription factor POU3F2 is expressed in human gliomas and promotes tumorigenesis in vivo. [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 2101. doi:10.1158/1538-7445.AM2015-2101
Abstract 2103: SOX2 suppresses PTEN expression via miR-19a in esophageal squamous cell carcinoma
Authors: Gen Yasuyuki , Yasui Kohichiroh , Kitaichi Tomoko , Dohi Osamu , Ito Yoshito
Published: August 2015

We previously showed that SOX2, a master regulator during embryogenesis, is an amplification target of 3q26.33 in esophageal squamous cell carcinoma (ESCC), and that SOX2 promotes ESCC proliferation in vitro and in vivo via activation of the AKT/mammalian target of rapamycin complex 1 (mTORC1) signaling pathway. However, the mechanisms by which SOX2 promotes AKT/mTORC1 signaling remain to be elucidated. We determined that SOX2 decreased PTEN, a negative regulator of AKT signaling, via microRNA (miRNA) in ESCC cells. SOX2 knockdown increased PTEN and decreased phosphorylated AKT (p-AKT), and PTEN knockdown restored p-AKT decreased by SOX2 knockdown in SOX2 amplified ESCC cells (KYSE70, KYSE140 and TE10 cells), indicating that SOX2 elevates p-AKT via PTEN suppression in these cells. DICER knockdown increased PTEN expression without SOX2 expression change in these cells, suggesting that PTEN expression is regulated by miRNA. Therefore we hypothesized that SOX2 may suppress PTEN expression through miRNA regulation. To examine whether SOX2 modulate the expression of miRNAs, we investigated the expression of cancer-related 84 miRNAs using miRNA PCR arrays, and found that SOX2 elevated miR-17∼92 cluster (miR-17, miR-18a, miR-19a, miR-19b-1, miR-20a, and miR-92) in KYSE70 cells. Quantitative RT-PCR confirmed that the expression of mir-17∼92 cluster is elevated by SOX2 in KYSE70, KYSE140, and TE10 cells. Of these miRNA, transfection of miR-19a mimic suppressed PTEN and conversely miR-19a inhibition elevated PTEN in these cells. Collectively, SOX2 may suppress PTEN expression via miR-19a in ESCC cells.

Citation Format: Yasuyuki Gen, Kohichiroh Yasui, Tomoko Kitaichi, Osamu Dohi, Yoshito Ito. SOX2 suppresses PTEN expression via miR-19a in esophageal squamous cell 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 2103. doi:10.1158/1538-7445.AM2015-2103
Abstract 2107: YAP protein expression and subcellular localization in pediatric liver tumors
Authors: LaQuaglia Michael J. , Grijalva James , Mueller Kaly , Perez-Atayde Antonio , Kim Heung Bae , Sadri-Vakili Ghazaleh , Vakili Khashayar
Published: August 2015

Purpose: Currently, there is no effective chemotherapy for the treatment of hepatocellular carcinoma (HCC). Both HCC and hepatoblastoma (HB) remain lethal in their metastatic forms. Therefore, understanding the molecular mechanisms of tumor survival and proliferation is critical for developing novel therapies. Our lab has shown that YAP, a transcription factor, is involved in liver regeneration. Specifically, increases in nuclear YAP have been coupled with expression of genes important for cell proliferation. We therefore sought to examine whether there is increased nuclear localization of YAP in liver tumors.

Methods: Tumor tissue and normal liver tissue from 3 pediatric patients with HCC (all males, mean age 10.63 years), and 6 HB patients (4 males and 2 females, mean age 2.99 years) were stained for YAP using immunohistochemistry. Multiple high-power fields were photographed for each sample. Using ImageJ software, the number of YAP-positive nuclei were counted and expressed as a mean percentage of total nuclei.

Results: The percentage of nuclei staining positive for YAP was increased in tumor cells compared to normal liver in 3/3 of the HCC tumors. In addition, YAP nuclear staining was significantly increased in 2/6 of the HB tumors.

Conclusion: All HCC tumors and 33% of the HB showed an increase in the nuclear localization of transcription factor YAP. The increased activity of YAP in the nuclei of tumor cells may indicate a crucial role for YAP in tumor proliferation and survival. Targeting YAP or its upstream pathway (Hippo) may provide a novel target for therapy.

Citation Format: Michael J. LaQuaglia, James Grijalva, Kaly Mueller, Antonio Perez-Atayde, Heung Bae Kim, Ghazaleh Sadri-Vakili, Khashayar Vakili. YAP protein expression and subcellular localization in pediatric liver tumors. [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 2107. doi:10.1158/1538-7445.AM2015-2107
Abstract 2112: A novel mechanism of NF-κB regulation in bladder cancer
Authors: Mukherjee Neelam , Ghosh Rita
Published: August 2015

Protein methyltransferases (PMTs) methylate protein lysine and arginine residues and play an important role in regulating chromatin remodeling and gene transcription. Deregulation of SET-domain-containing PMTs has a crucial role in carcinogenesis (1). SETD6 is a novel methyltransferase that has been shown to monomethylate p65/RelA (a principal subunit of NF-κB) at Lys310. Monomethylated p65 recruits GLP (G9A-like protein), a histone dimethyl transferase to the NF-κB target genes, which results in transcriptional repression (2). The role of SETD6, in carcinogenesis in general and bladder cancer in particular is unknown. We identified SETD6 as a significantly upregulated gene in a screen of chromatin modifying genes that are differentially regulated in bladder cancer cells compared with normal urothelial cells. Subsequently qPCR and immunoblotting showed upregulation of SETD6 at the mRNA and protein level in bladder cancer cells and tissues relative to their normal counterparts. Surprisingly, we found that overexpression of SETD6 increased nuclear level of NF-κB, phosphorylation of NF-κB at Ser536 and the transcriptional activity of NF-κB. Overexpression of wild type SETD6 increased viability and proliferation of non-tumorigenic urothelial cells while overexpression of mutant SETD6 had no effect. Furthermore, knocking down SETD6 transiently with si-SETD6 significantly decreased viability and proliferation of bladder cancer cells suggesting that SETD6 may have a growth promoting role in bladder cancer. Our data shows for the first time that non-histone protein lysine methylation of NF-κB-p65 by SETD6 may be a novel way to regulate NF-κB pathway in bladder cancer. The lack of curative strategies for bladder cancer leads to recurrence rates of 45-70% with >15% of patients developing muscle invasive disease (3). These observations underscore the need to understand survival mechanism in bladder cancer. In this regard our findings are highly significant and could lead to targeting SETD6/ NF-κB axis in bladder cancer in the future.

References:

1.

Schneider, R., et al. (2002) Unsafe SETs: histone lysine methyltransferases and cancer. Trends in biochemical sciences, 27, 396-402.

2.

Levy, D., et al. (2011) Lysine methylation of the NF-κB subunit RelA by SETD6 couples activity of the histone methyltransferase GLP at chromatin to tonic repression of NF-κB signaling. Nature immunology, 12, 29-36.

3.

Nativ, O., et al. (2009) Combined thermo-chemotherapy for recurrent bladder cancer after bacillus Calmette-Guerin. The Journal of urology, 182, 1313-1317.

Citation Format: Neelam Mukherjee, Rita Ghosh. A novel mechanism of NF-κB regulation in bladder 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 2112. doi:10.1158/1538-7445.AM2015-2112
Abstract 2124: Analysis of the functional relevance of novel alternative splicing events in non-small cell lung cancer
Authors: de Miguel Fernando J. , Sharma Ravi D. , Reclusa Pablo , Pajares María J. , Rubio Angel , Pio Ruben , Montuenga Luis M.
Published: August 2015

Lung cancer is the most common cause of death by cancer worldwide. Deregulation of alternative splicing (AS) is becoming increasingly relevant for the understanding of the molecular mechanisms of lung tumor progression. Genome-wide microarrays containing probes in exons and junctions can be used to identify differential AS events. We developed an algorithm, called ExonPointer, which was optimized to detect AS events using this kind of platforms. Using this technique, we successfully detected splicing events regulated by the oncogene serine/arginine-rich splicing factor 1 (SRSF1) in lung cancer cell lines. Moreover, four of these genes, ATP11C, IQCB1, TUBD1 and PRRC2C, showed a significantly different pattern of AS in primary non-small cell lung cancer (NSCLC) compared with normal lung tissue. In the case of PRRC2C, we also demonstrated that the deregulation of the splicing event was important for tumor progression. We next applied the same technique to target differential AS events in NSCLC primary tissues (13 lung squamous cell carcinomas and 9 lung adenocarcinomas). Their paired normal lung tissues were used as reference. The validation rate for the top 20 events identified by ExonPointer was 70%. Gene cluster analyses showed an enrichment of clusters implicated in cancer development such as Cellular Growth and Proliferation, or Cell Death and Survival. Among others, we observed accumulation of a tumor-specific splice variant in Mitogen-Activated Protein Kinase 9 (MAPK9 or JNK2). The tumor-specific variant for this gene contains an alternative exon 2 which causes a frame shift introducing a premature stop codon. Functional analyses using isoform-specific siRNAs showed that this variant did not affect tumor growth or progression. However, its expression significantly correlated with the expression of Regulator of Nonsense Transcripts 3A and B (UPF3A and UPF3B) in 42 lung cancer cell lines. These proteins belong to the Nonsense Mediated Decay (NMD) pathway, which eliminates aberrant mRNA transcripts, suggesting that the presence of some aberrant AS variants in lung cancer are caused by a malfunction of NMD. In conclusion, we have developed a reliable tool for the high throughput analysis of AS, and have identified new splicing events in lung cancer that can be studied as potential diagnostic markers and therapeutic targets.

Citation Format: Fernando J. de Miguel, Ravi D. Sharma, Pablo Reclusa, María J. Pajares, Angel Rubio, Ruben Pio, Luis M. Montuenga. Analysis of the functional relevance of novel alternative splicing events in non-small cell lung 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 2124. doi:10.1158/1538-7445.AM2015-2124
Abstract 2126: Comprehensive transcriptome analysis reveals that nonsense-mediated mRNA decay is not globally suppressed in lung adenocarcinomas
Authors: Kong Xiangyin
Published: August 2015

As a well-known mRNA surveillance mechanism, the nonsense-mediated mRNA decay (NMD) pathway ensures the rapid degradation of mRNAs containing premature termination codons (PTC). Prior research has shown that features of the tumor microenvironment, such as hypoxia and amino acid deprivation, might inhibit NMD activity. However, a systemic analysis of NMD in cancer is seldom performed. Rather than focusing on only a few NMD targets and using artificial interventions as in previous experiments, we aimed to analyze NMD in cancer from a global perspective. By analyzing lung Adenocarcinomas in detail, we observed comparable decay efficiencies in tumors for both annotated and predicted NMD targets. Interestingly, the expression patterns of key NMD factors are altered, indicating that cancer cells might prefer to use different NMD degradation pathways. We also identified a subset of NMD targets with unique manners in either tumor or adjacent tissues. Besides, our analyses further demonstrate that the NMD efficiencies vary across tumors, supporting the heterozygosity of lung Adenocarcinomas.

Citation Format: Xiangyin Kong. Comprehensive transcriptome analysis reveals that nonsense-mediated mRNA decay is not globally suppressed in lung adenocarcinomas. [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 2126. doi:10.1158/1538-7445.AM2015-2126
Abstract 2127: HnRNP Q1 contributes to tumorigenesis through the translational regulation of Aurora-A in colorectal cancer
Authors: Hung Liang-Yi
Published: August 2015

HnRNP Q1 is a RNA-binding protein which belongs to hnRNP Q family and involves in pre-mRNA splicing, mRNA transport, translation and turnover. Data from Oncomine, which is a cancer microarray database, indicates that hnRNP Q1 is overexpressed in colorectal cancer. According the literatures, hnRNP Q1 can increase the translational efficiency through binding to the 5′-untranslated region (5′-UTR) of target genes. From RNA-IP assay following the next generation sequencing (NGS), we found that hnRNP Q1 regulates a group of cell cycle-related genes. Among them, Aurora-A is one of the targets of hnRNP Q1. Aurora-A is a cell-cycle dependent kinase involved in regulating centrosome duplication, maturation, and spindle assembly during mitosis. Abnormally elevated Aurora-A will cause mitotic defect and aneuploidy, which might lead to genomic instability and tumorigenesis. In addition, overexpression of hnRNP Q1 is positively correlated with overexpressed Aurora-A in clinical colorectal cancer tissues. Our previous report demonstrated that Aurora-A can be translational up-regulated in colorectal cancer. In this study, we demonstrated that hnRNP Q1 can bind to Aurora-A 5′-UTRs and also mapped the interacting domain between Aurora-A 5′UTR and hnRNP Q1 has been mapped by Biacore analysis, RNA-IP and biotin pull-down assay. Overexpressed hnRNP Q1 enhances the Aurora-A protein expression but doesn't alter its mRNA level. In contrast, repressed expression of hnRNP Q1 decreases the translation of Aurora-A. Importantly, we found that Aurora-A 5′UTR has IRES-dependent translational activity in a cell cycle regulated manner. Overexpression of hnRNP Q1 can up-regulate the IRES-dependent translation of Aurora-A during cell cycle progression, and promotes the cells proliferation. Taken together, our data indicate hnRNP Q1 is a novel trans-acting factor that binds on the Aurora-A 5′-UTRs and regulates it's translation, which may increase the cell proliferation and contribute to the tumorigenesis of colorectal cancer.

Citation Format: Liang-Yi Hung. HnRNP Q1 contributes to tumorigenesis through the translational regulation of Aurora-A in colorectal 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 2127. doi:10.1158/1538-7445.AM2015-2127
Abstract 2131: The Arkadia-ESRP2 axis suppresses tumor progression: Analyses in clear cell renal cell carcinoma
Authors: Mizutani Anna , Koinuma Daizo , Seimiya Hiroyuki , Miyazono Kohei
Published: August 2015

Tumor-specific alternative splicing is implicated in the progression of cancer, including clear cell renal cell carcinoma (ccRCC), a major subtype of kidney cancer accounting for 70-80% of all kidney cancer patients. Using ccRCC RNA-sequencing data from The Cancer Genome Atlas, we found that epithelial splicing regulatory protein 2 (ESRP2), one of the key regulators of alternative splicing in epithelial cells, is expressed in ccRCC, while ESRP1 is decreased in most of the ccRCC as previously reported. ESRP2 mRNA expression did not correlate with the overall survival rate of ccRCC patients, but the expression of some ESRP-target exons, including ENAH exon 11a, ITGA6 exon 27, and SLK exon 13, correlated with the good prognosis and with the expression of Arkadia (also known as RNF111) in ccRCC. Arkadia, a RING-type E3 ligase, that is known to enhance TGF-β signaling activities, physically interacted with ESRP2, induced polyubiquitination, and modulated its splicing function. Arkadia and ESRP2 suppressed ccRCC tumor growth in a coordinated manner. Lower expression of Arkadia correlated with advanced tumor stages and poor outcomes in ccRCC patients. This study thus reveals a novel tumor-suppressive role of the Arkadia-ESRP2 axis in ccRCC.

Citation Format: Anna Mizutani, Daizo Koinuma, Hiroyuki Seimiya, Kohei Miyazono. The Arkadia-ESRP2 axis suppresses tumor progression: Analyses in clear cell renal cell 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 2131. doi:10.1158/1538-7445.AM2015-2131
Abstract 2133: Investigating the molecular interaction between KRAS mRNA and RNA binding protein CRD-BP
Authors: Mackedenski Sebastian J. , Lee Chow H.
Published: August 2015

The Coding Region Determinant-Binding Protein (CRD-BP) is an RNA-binding protein which, among other functions, provides a protective effect for select target mRNA transcripts against degradation, effectively increasing their half-life leading to elevated level of gene expression. CRD-BP was first identified due to its inherent ability to bind to a specific coding region of c-myc mRNA. It has further been demonstrated to convey a protective effect to target transcripts, including GLI1, MITF, CD44, and KRAS as well as a host of other mRNAs. CRD-BP is normally under strict spatiotemporal regulation; expressed only during early developmental stages where rapid growth is necessary. However, CRD-BP re-expression has been reported to occur in a number of human cancers, suggesting its roles as an onco-protein. Abnormal expression or function of KRAS, a CRD-BP target, is implicated in some cancers; roughly 90% of pancreatic cancers, 60% of colorectal, and 50% of lung cancers, making KRAS an attractive target for cancer therapies and highlighting the significance of understanding the CRD-BP-KRAS mRNA interaction. To help understand the CRD-BP-RNA interaction, we have previously mutated the first glycine of the G-X-X-G motif in the KH domains of CRD-BP to an aspartate. Using electrophoretic mobility shift assay, we showed that mutation at any two KH domains, except the KH3 and KH4 di-domains, completely abrogated CRD-BP-c-myc and CD44 RNAs interactions. To understand the CRD-BP-KRAS RNA interaction, we used the identical site-directed KH mutants of CRD-BP to assess their binding profiles to a 185-nt KRAS RNA corresponding to a specific coding region of KRAS mRNA. Using EMSA, additional binding assessments were carried out in a systematic fashion to determine the smallest region of KRAS mRNA bound by CRD-BP. In addition, plasmids carrying these variants of FLAG-CRD-BP were transfected into HeLa cells. Immuno-precipitation (IP) using anti-FLAG was performed, followed by RT-qPCR to measure the relative amounts of KRAS mRNA associated with each of the variants as compared to the controls. The in vitro EMSA and IP cell data were all consistent with the notion that all KH domain(s) are critical for CRD-BP-KRAS RNA interaction. These experiments and their results will be discussed in further detail upon presentation.

Citation Format: Sebastian J. Mackedenski, Chow H. Lee. Investigating the molecular interaction between KRAS mRNA and RNA binding protein CRD-BP. [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 2133. doi:10.1158/1538-7445.AM2015-2133
Abstract 214: Characterization of the near-infrared fluorescent protein iRFP for plate-based assays and animal imaging
Authors: Gong Haibiao , Kovar Joy L. , Urlacher Teresa
Published: August 2015

Fluorescence imaging has been extensively used in biological research because of its high sensitivity, signal stability, and simplicity of multiplexing. Compared to fluorophores in the visible spectral region, fluorophores with excitation (Ex) and emission (Em) maxima in the near-infrared (NIR) region (700-900 nm) are desirable in many applications. Cells, buffers and plastic materials generate less background fluorescence in the NIR region. As a result, NIR fluorescence imaging offers higher sensitivity and better signal-to-background ratio (SBR) compared to visible fluorescence. In addition, due to the reduced light absorption and scattering in animal tissues, NIR fluorescence is particularly suitable for in vivo imaging studies. In this study, we characterized the NIR fluorescent protein iRFP for plate-based assays and animal imaging. HEK293 cells stably expressing iRFP were established and designated as HEK293-iRFP. Serial dilutions of HEK293-iRFP cells were seeded in 96-well plate and scanned on the Odyssey CLx Imaging System. A good linear relationship between fluorescence signal intensity and cell number was obtained. The lower limit-of-detection was determined as 763 cells. To evaluate the performance of iRFP for animal imaging, the iRFP-expressing plasmid was delivered into mouse liver cells using a hydrodynamic injection method. A dose- and time-dependent pattern of fluorescence signal was observed after plasmid injection. At 8 h post-injection of 10 μg of iRFP plasmid, a SBR of 41 was observed by live animal imaging. Analysis of liver tissue lysates revealed a SBR of 148. Other tissues, including kidney, spleen, heart and lung, showed very little fluorescence signal. Our results demonstrate that iRFP is a good reporter for both plate-based assays and animal imaging studies.

Citation Format: Haibiao Gong, Joy L. Kovar, Teresa Urlacher. Characterization of the near-infrared fluorescent protein iRFP for plate-based assays and animal imaging. [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 214. doi:10.1158/1538-7445.AM2015-214
Abstract 2144: Kinetic mechanism of the lysine methyltransferase SMYD3 using MAP3K2 protein substrate
Authors: Riera Thomas V. , Wigle Tim J. , Gureasko Jodi , Boriack-Sjodin P. Ann , Copeland Robert A.
Published: August 2015

Dysregulation of DNA and histone methylation has been shown to play fundamental roles in carcinogenesis. Recently however, protein methylation in the cytoplasm has been linked to oncogenic Ras signaling. Mazur and colleagues demonstrated that SMYD3 (SET and MYND domain containing protein 3), a primarily cytoplasmic protein methyltransferase, can methylate MAP3K2 using S-adenosyl-L-methionine (SAM) as a cofactor (Mazur et al. 2014. Nature 510(7504):283-7). Methylation by SMYD3 preserves the active, phosphorylated state of MAP3K2 sustaining activation of the Ras/Raf/MEK/ERK/ signaling module and potentiating formation of Ras-driven cancers. Here, we characterized the kinetic mechanism of SMYD3 using MAP3K2 protein as a substrate. SMYD3 activity was measured with a radiometric assay using 3H-SAM. Initial velocity, product and dead-end inhibitor studies indicate that SMYD3 uses a random kinetic mechanism where both SAM and MAP3K2 can bind apo-SMYD3. This was supported by substrate and ligand binding assays using SPR. Together, these studies elucidate the kinetic mechanism of SMYD3 with MAP3K2 and provide a context for inhibitor development.

Citation Format: Thomas V. Riera, Tim J. Wigle, Jodi Gureasko, P. Ann Boriack-Sjodin, Robert A. Copeland. Kinetic mechanism of the lysine methyltransferase SMYD3 using MAP3K2 protein substrate. [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 2144. doi:10.1158/1538-7445.AM2015-2144
Abstract 2154: ERK/MAPK pathway inhibits tumorigenesis and cellular reprogramming of pancreatic cancer cells
Authors: le Calvé Benjamin , Deschenes-Ximard Xavier , Kottakis Filippos , Bourdeau Véronique , Lessard Frédéric , Moineau-Vallée Karine , Saint-Germain Emmanuelle , Fitamant Julien , Perera Rushika Miriam , Bardeesy Nabeel , Ferbeyre Gerardo
Published: August 2015

Reprogrammation of cancer cells into stem-like state is one the most important mechanisms implicated in tumor initiation, metastasis and resistance to chemotherapies. Acquisition of stem-like cell property is directly correlated with the level of activation of the ERK/MAPK pathway. Our study focuses on the implication of the downregulation of this pathway during the transition of pancreatic benign neoplasms to pancreatic ductal adenocarcinoma. This transformation is directly correlated with the acquisition of stem-like cells properties. Conversely, hyperactivation of the ERK pathway using phosphatase inhibitors abrogates the stem-like cell phenotype.

We analyzed the variation of genes expression by microarrays analysis between cells obtained from pancreatic intraepithelial neoplasias and pancreatic cancer. These analyses implicate the expression of DUSPs and genes related to stem cell biology in tumor progression during pancreatic cancer.

Citation Format: Benjamin le Calvé, Xavier Deschenes-Ximard, Filippos Kottakis, Véronique Bourdeau, Frédéric Lessard, Karine Moineau-Vallée, Emmanuelle Saint-Germain, Julien Fitamant, Rushika Miriam Perera, Nabeel Bardeesy, Gerardo Ferbeyre. ERK/MAPK pathway inhibits tumorigenesis and cellular reprogramming of pancreatic cancer cells. [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 2154. doi:10.1158/1538-7445.AM2015-2154
Abstract 2158: PDK1-dependent activation of RSK is an absolute requirement for PI3K oncogenic activity in the thyroid gland
Authors: Orlacchio Arturo , Di Cristofano Antonio
Published: August 2015

Aberrant activation of the phosphatidylinositol-3 kinase (PI3K)/Akt pathway plays a key role in thyroid tumorigenesis, particularly in follicular thyroid cancer (FTC) and anaplastic thyroid cancer (ATC). Accordingly, conditional deletion of Pten in the mouse thyroid epithelium induces constitutive activation of the PI3K/AKT pathway, and causes thyroid hyperplasia at birth that progresses to invasive and metastatic follicular carcinoma. This phenotype is dramatically accelerated by simultaneous deletion of Trp53, leading to the development of ATC, or activation of Kras, leading to poorly differentiated carcinomas.

The PDK1 kinase plays a central role in the PI3K signaling cascade, acting as a master regulator of PI3K downstream effectors belonging to the AGC kinases family, such as AKT and S6K1. Additional AGC kinases are controlled by PDK1 independent of PI3K activity.

PDK1 possesses a substrate docking site, the “PIF pocket”, required for the phosphorylation of most AGC kinases (including S6K, PKC, SGK, and RSK) but not of AKT. The L155E PDK1 mutant disrupts the PIF pocket and impairs AGC kinase activation, without any effect on AKT activation.

Thanks to this differential mechanism of substrate activation, we are defining the relative contribution of AKT and other PDK1 targets to the neoplastic transformation process in Pten-/- thyrocytes.

Surprisingly, in vivo disruption of the PIF-pocket completely abrogates neoplastic transformation of Pten-/- thyroids, even in the face of AKT constitutive activation. This finding supports the hypothesis that the linear PI3K-AKT pathway is not sufficient to drive thyroid transformation and that concomitant activation of additional PDK1-dependent pathways is absolutely required.

We now show that RSK family members (RSK1-3) are essential components of this PDK1-dependent accessory pathway. Remarkably, RSK3 levels in the thyroid are increased in the absence of Pten, and RSK activity in thyroid cells is abrogated when PDK1 PIF-pocket is mutated.

Genetic and pharmacological inhibition of RSK drastically impairs [Pten,Trp53]-/- ATC cell proliferation both in vitro and in vivo, in syngeneic allograft models.

Taken together, our data identify a new signaling cascade that is essential for neoplastic transformation of thyroid epithelial cells, and thus a new critical candidate for drug development.

Citation Format: Arturo Orlacchio, Antonio Di Cristofano. PDK1-dependent activation of RSK is an absolute requirement for PI3K oncogenic activity in the thyroid gland. [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 2158. doi:10.1158/1538-7445.AM2015-2158