bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1910: Tumor-suppressive microRNAs (miR-26a/b, miR-29a/b/c and miR-218) concertedly regulate metastasis-promoting LOXL2 in prostate cancer
Authors: Kato Mayuko , Kurozumi Akira , Goto Yusuke , Matsushita Ryosuke , Fukumoto Ichiro , Okato Atsushi , Nishikawa Rika , Sakamoto Shinichi , Ichikawa Tomohiko , Seki Naohiko
Published: July 2016

BACKGROUNDS:

Most patients with prostate cancer (PCa) initially respond to androgen-deprivation therapy, but eventually becomes hormone-insensitive and progress to castration-resistant prostate cancer (CRPC). CRPC is difficult to treat, and most clinical trials for advanced PCa have shown limited benefits, with disease progression and metastasis to the skeleton or other sites. Therefore, understanding the molecular mechanisms of CRPC and the metastatic pathways underlying PCa using genomic approaches would help to prevent and improve therapies for the disease. Our previous studies showed that three tumor-suppressive microRNAs (miRNAs), miR-26a/b, miR-29a/b/c and miR-218, significantly inhibited cancer cell migration and invasion. Therefore, we hypothesized that these miRNAs-regulated target genes deeply contributed to cancer metastasis.

METHODS:

Genome-wide gene expression analysis and in silico analysis were applied to investigate molecular targets regulated by miR-26a/b, miR-29a/b/c and miR-218 in PCa cells. A luciferase reporter assay was carried out to determine whether 3’ UTR of target genes have actual biding sites for these miRNAs. To investigate the functional role of target genes in PCa cells, loss-of-function studies by using siRNAs of targets genes were performed.

RESULTS:

Our in silico analysis and luciferase reporter assays showed that lysyl oxidase-like 2 (LOXL2) was directly regulated by tumor-suppressive miR-26a/b, miR-29a/b/c and miR-218 in PCa cells. Overexpression of LOXL2 was observed in PCa clinical specimens by immunohistochemical staining. Moreover, downregulating the LOXL2 gene significantly inhibited cell migration and invasion in PCa cells. The function of the LOXL2 is covalent crosslinking of collagen and/or elastin in the ECM. Aberrant expression of LOXL2 has been reported in several diseases, including cancers.

CONCLUSIONS:

Our present data showed that the gene promoting metastasis, LOXL2, was epigenetically regulated by tumor-suppressive microRNAs, miR-26a/b, miR-29a/b/c and miR-218 providing important insights into the molecular mechanisms of PCa metastasis. Elucidation of tumor-suppressive miRNAs regulated molecular pathways and targets could provide new information on potential therapeutic strategies in PCa.

Citation Format: Mayuko Kato, Akira Kurozumi, Yusuke Goto, Ryosuke Matsushita, Ichiro Fukumoto, Atsushi Okato, Rika Nishikawa, Shinichi Sakamoto, Tomohiko Ichikawa, Naohiko Seki. Tumor-suppressive microRNAs (miR-26a/b, miR-29a/b/c and miR-218) concertedly regulate metastasis-promoting LOXL2 in prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1910.
Abstract 1913: Inhibition of S-Adenosylmethionine-dependent methyltransferase resists TGF-β1-induced EMT of pancreatic cancer via microRNA alterations
Authors: Govindarajan Rajgopal , Modi Hardik
Published: July 2016

Identification of epigenetic reversal agents for use in combination chemotherapies for human pancreatic ductal adenocarcinomas remains an unmet clinical need. Pharmacological inhibitors of EZH2 are emerging as potential epigenetic reversal agents in various solid tumors and leukemia, however, the surprisingly small set of mRNA transcripts identified with EZH2 knockdown suggests novel mechanisms to govern their anti-tumorigenic effects. Here, we report 3-deazaneplanocin-A, an inhibitor of S-adenosyl-L-homocysteine hydrolase and EZH2 histone lysine-N-methyltransferase, to significantly reprogram the noncoding miRNA expression, including a subset of PDAC-downregulated miRNAs, to dampen TGF-β-induced EMT signals in pancreatic cancer. Particularly, we identify epigenetic silencing of miR-663a and -4787-5p in PDAC tissues and cell lines that are reactivated by 3-deazaneplanocin-A to directly target TGF-β1 for RNA interference. Lentiviral overexpression of miR-663a and -4787-5p partially phenocopied 3-deazaneplanocin-A's EMT resisting effects in pancreatic cancer cells, whereas locked nucleic acid antimiR-663a and -4787-5p mitigated them. In vivo, 3-deazaneplanocin-A, miR-663a and -4787-5p significantly reduced tumor burden and metastasis in an orthotopic mouse pancreatic tumor model with miR-663a displaying more potency than miR-4787-5p in curtailing TGF-β1 signaling pathway. These findings reveal tumor miRNA reprogramming as a novel approach for controlling TGF-β-induced EMT in human PDAC and uncover a list of putative miRNA targets that may guide further selection and development of synthetic histone methylation reversal agents for clinical use.

Citation Format: Rajgopal Govindarajan, Hardik Modi. Inhibition of S-Adenosylmethionine-dependent methyltransferase resists TGF-β1-induced EMT of pancreatic cancer via microRNA alterations. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1913.
Abstract 1915: microRNA-320a inhibits cancer cell migration and invasion through targeting LAMP1 in castration-resistant prostate cancer
Authors: Okato Atsushi , Goto Yusuke , Kurozumi Akira , Kato Mayuko , Matsushita Ryosuke , Kojima Satoko , Sakamoto Shinichi , Ichikawa Tomohiko , Seki Naohiko
Published: July 2016

BACKGROUND:

In early-stage prostate cancer (PCa), more than 90% of patients initially respond to the therapeutic use of androgen deprivation; however, many cases become refractory and progress to castration resistant prostate cancer (CRPC). CRPC is difficult to treat, and most clinical trials for advanced PCa with metastasis to bones or other sites have shown limited benefits. Therefore, understanding the molecular mechanisms of metastatic pathways of CRPC underlying current genomic approaches would help to improve therapies for this disease.

Our recent studies of microRNA (miRNA) expression signatures of CRPC revealed that microRNA-320a (miR-320a) was significantly reduced in cancer tissues, suggesting miR-320a acts as a tumor-suppressor in CRPC cells. The aim of this study was to investigate the functional significance of miR-320a in CRPC cells and to identify miR-320a-mediated target genes involved in oncogenesis and metastasis of the disease.

METHODS:

Gain-of-function studies were performed using transfection of mature miR-320a into cancer cells (DU145 and PC3). Genome-wide gene expression analysis and in silico analysis were applied to identify molecular targets regulated by miR-320a in PCa cells. A luciferase reporter assay was carried out to determine regulation of miR-320a. Loss-of-function studies using siRNA were performed to investigate the functional role of target genes.

RESULTS:

The expression levels of miR-320a were significantly reduced in PCa tissues and the cell lines. Restoration of mature miR-320a in cancer cells led to significant inhibition of cell migration and invasion activities in PCa cell lines. Lysosomal-associated membrane protein 1 (LAMP1) was identified as a direct target of miR-320a in this assays. Silencing of LAMP1 significantly inhibited cell proliferation, migration and invasion in PCa cells. Moreover, overexpression of LAMP1 was observed in PCa and CRPC clinical specimens.

CONCLUSIONS:

To the best of our knowledge, this is the first report demonstrating that tumor-suppressive miR-320a directly regulated LAMP1 in PCa cells. The identification of novel target gene regulated by tumor-suppressive miRNAs may lead to a better understanding of molecular mechanisms of CRPC metastatic pathways.

Citation Format: Atsushi Okato, Yusuke Goto, Akira Kurozumi, Mayuko Kato, Ryosuke Matsushita, Satoko Kojima, Shinichi Sakamoto, Tomohiko Ichikawa, Naohiko Seki. microRNA-320a inhibits cancer cell migration and invasion through targeting LAMP1 in castration-resistant prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1915.
Abstract 1916: Targeting a MAPK-miR-124-Sox9 axis radiosensitizes human glioblastoma cells
Authors: Sabelstrom Hanna , Jandial Rahul , Shchors Ksenya , Masic Selma , Yuan Edith , Fenster Trenten , Saxena Supna , Ho Allen , Nicolaides Theodore P. , Wong Robyn , Yakovenko Stanislava , Berger Mitchel H. , Snyder Evan Y. , Jakobsson Johan , Weiss William A. , Persson Anders I.
Published: July 2016

Glioblastoma (GBM) is the most common and aggressive malignant primary brain tumor. Although genetic alterations and overexpression of genes in the RTK/RAS/MEK/ERK pathway (MAPK) is known to drive GBM aggressiveness, less is known about down-stream effector genes. We confirmed that EGFR amplified human GBMs display high levels of the transcription factor SOX9, a known barrier to neurogenesis and target of microRNA-124 (miR-124). Surprisingly, a subpopulation of SOX9-negative tumor cells expressed markers of mature neurons. Here, we demonstrate that the MEK inhibitor PD325901 depletes SOX9, reduces proliferation, and induces neurogenesis in a mouse GBM model and human GBM cells. We find that MAPK signaling regulates a large cluster of miRNAs in mouse neurosphere cultures, including miR-124. In addition, PD325901 effectively induced miR-124 levels in SOX9-expressing human GBMs. Using a doxycycline-regulable approach, we demonstrate that miR-124 overexpression depletes SOX9 levels and induces neurogenesis in a concentration- and time-dependent manner in human GBM tumorsphere cultures. Doxycycline treatment of athymic nude mice intracranially xenografted with human GBM cells resulted in reduced tumor growth, robust neuronal differentiation, and increased overall survival. Finally, we demonstrate that a brief 3-day treatment with PD325901 or doxycycline effectively radiosensitizes human GBM cells and extends overall survival of xenografted mice. Our work shows that the miR-124-SOX9 axis is a down-stream effector pathway that drives GBM aggressiveness. Long-term exposure to PD325901 is associated with adverse side-effects in patients. We propose that a priming regimen of the clinically relevant MEK inhibitor PD325901 should radiosensitize SOX9-expressing tumors in patients and result in reduced side-effects compared to continuous treatment protocols.

Citation Format: Hanna Sabelstrom, Rahul Jandial, Ksenya Shchors, Selma Masic, Edith Yuan, Trenten Fenster, Supna Saxena, Allen Ho, Theodore P. Nicolaides, Robyn Wong, Stanislava Yakovenko, Mitchel H. Berger, Evan Y. Snyder, Johan Jakobsson, William A. Weiss, Anders I. Persson. Targeting a MAPK-miR-124-Sox9 axis radiosensitizes human glioblastoma cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1916.
Abstract 1922: miR-301 expression is deregulated in rhabdomyosarcoma
Authors: Rota Rossella , Gualtieri Alberto , Cossetti Cristina , Pomella Silvia , Adesso Laura , Locatelli Franco
Published: July 2016

Rhabdomyosarcoma (RMS) is an aggressive pediatric soft tissue sarcoma of myogenic derivation. Conversely to normal myoblasts, RMS cells are unable to differentiate, thus they proliferate indefinitely. MicroRNAs (miRNAs) are short (20-24 nt) non-coding RNAs that are involved in post-transcriptional regulation of gene expression by affecting both the stability and translation of mRNAs. They are fundamental regulators of myogenesis and their deregulation has been involved in the pathogenesis of RMS. We evaluate here the expression and regulation of miR-301, which has been related to tumorigenesis in other types of cancers. We report here that the expression of miR-301 is upregulated in primary samples of both the alveolar RMS, which bears the fusion protein PAX3-FOXO1 (P3F) (ARMS), and the fusion-negative embryonal RMS (ERMS) compared to normal muscle tissues. Similarly, it appears higher in RMS cell lines versus normal skeletal myoblasts. Using an inhibitor of miR-301 (CONTRAmiR) we demonstrate that this miRNA favors RMS cell mobility and down-regulates p21Cip1, PTEN and DICER1 levels. However, the inhibition of miR-301 with the CONTRAmiR does not reduce tumor cell proliferation suggesting that the regulation of cell cycle is not under the control of this miRNA in RMS. Interestingly, the expression of SKA2 (Spindle And Kinetochore Associated Complex Subunit 2), whose gene hosts miR-301, is also upregulated in our setting. Experiments are ongoing to determine whether miR-301 and SKA2 are associated with RMS pathogenesis and could be targets for an anti-RMS therapy.

This work was supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC).

Citation Format: Rossella Rota, Alberto Gualtieri, Cristina Cossetti, Silvia Pomella, Laura Adesso, Franco Locatelli. miR-301 expression is deregulated in rhabdomyosarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1922.
Abstract 1924: miR-142-3p is a candidate mediator of chloride intracellular channel 4 (CLIC4) loss during squamous cancer progression
Authors: Carofino Brandi L. , Yuspa Stuart H.
Published: July 2016

Chloride intracellular channel 4 (CLIC4) is a broadly expressed protein that has been implicated in multiple cellular processes, including cellular differentiation and apoptosis. As a soluble factor, CLIC4 translocates to the nucleus following exposure to a variety of stimuli, including TGF-β, TNF-α, and etoposide. Nuclear CLIC4 potentiates TGF-β signaling by preventing the dephosphorylation of phospho-SMAD2 and 3 and promotes growth arrest. Although its primary function is as a tumor suppressor, CLIC4 expression is progressively diminished during cancer progression and CLIC4 protein is excluded from the nucleus of many human epithelial neoplasms and squamous cell carcinoma (SCC) cell lines. To date, no gene deletions or mutations have been identified to account for this phenomenon; thus, identifying the mechanism of CLIC4 loss is our primary focus. To determine if epigenetic gene silencing is responsible, we evaluated the methylation status of the CLIC4 promoter in a progression panel (normal, papilloma, SCC) of epithelial cell lines by performing bisulfite sequencing. No differential promoter methylation was found between the cell lines, suggesting that the regulation may be post-transcriptional or post-translational. Because microRNAs (miRNAs) are known to regulate gene expression by mediating the degradation or translational repression of target mRNAs, we performed a bioinformatic analysis of the CLIC4 3’ UTR, which revealed a large number of putative targeting miRNAs. One such miRNA, miR-142-3p, has been predicted to target CLIC4 by multiple algorithms and has been shown to interact with the CLIC4 3’ UTR by high-throughput sequencing and crosslinking immunoprecipitation. We confirmed the functional activity of miR-142-3p against the CLIC4 3’ UTR by using a 3’ UTR luciferase reporter assay and a miR-142-3p mimic. miR-142-3p is high in the tumor tissue and plasma of patients with head and neck SCC and is correlated with worse prognosis, where CLIC4 expression is known to be low. Thus, miR-142-3p may be responsible for mediating CLIC4 loss in squamous lesions and warrants further investigation.

Citation Format: Brandi L. Carofino, Stuart H. Yuspa. miR-142-3p is a candidate mediator of chloride intracellular channel 4 (CLIC4) loss during squamous cancer progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1924.
Abstract 1927: Myc-induced suppression of the RNA-binding protein ZAR2 in breast cancer cells
Authors: Misra Smita , Chaudhuri Gautam
Published: July 2016

Our objective is to verify the hypothesis that c-Myc-regulated oncomiR miR-130a-5p suppresses the level of the RNA-binding tumor suppressor protein ZAR2 in the cMyc-high aggressive breast cancer cells. We experimentally validated the binding of miR130a-5p miRISC to ZAR2 mRNA in vivo and inhibition of the translation of this mRNA by this binding. We used three commercially available c-Myc-high ZAR2-low breast cancer cell lines BT549, MDA-MB-453 and MDA-MB-231 and two c-Myc-low ZAR2-high breast cancer cells MCF7 and HCC38 for our studies. We cloned the full length 3′-UTR (179 bp) of human ZAR2 mRNA immediately downstream of the firefly luciferase open reading frame sequence contained in the reporter plasmid. We also have created different mutants of the 3’-UTR, each having mutation in each of the putative miR130a-5p binding sites. We transiently transfected the recombinant plasmid and miR130a-5p miRNA mimic into breast cancer cells and assessed luciferase activity or fluorescence 24-48 hours after transfection. We also have transfected just the reporter construct into breast cancer cells which express the endogenous miR130a-5p miRNAs, along with vectors which express mutant versions of the miRNA binding sites. In this experimental system the wild-type reporters had less activity than their respective mutants. To evaluate the functional importance of miR130a-5p miRNA/ZAR2 mRNA pair, we transiently over-expressed a miR130a-5p miRNA mimic in the cMyc-low breast cancer breast cancer cells and subsequently analyzed the level of ZAR2 protein levels. Our data showed that ZAR2 protein levels are decreased by the mimic increasing the in vitro invasiveness of these cells. Our results thus reveal a novel mechanism for the induction of aggressiveness by cMyc in the breast cancer cells. This research is supported in part by DOD-CDMRP IDEA Expansion Grant# BC103645 and NIH/NCI grant 1R21CA181920-01 to GC and NIH grant 1U54RR026140 TO SM

Citation Format: Smita Misra, Gautam Chaudhuri. Myc-induced suppression of the RNA-binding protein ZAR2 in breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1927.
Abstract 1929: The novel role of microRNA-128 in proneural to mesenchymal subtype transition in glioblastoma stem cells by targeting components of pro-oncogenic Polycomb Repressor Complex
Authors: Rooj Arun K. , Mineo Marco , Ricklefs Franz , Bronisz Agnieszka , Chiocca Ennio , Godlewski Jakub
Published: July 2016

Heterogeneous glioblastoma multiforme (GBM) was categorized based on transcriptional signatures into four subtypes (proneural (PN), neural, classical, and mesenchymal (MES)). In order to develop effective targeted therapeutic strategies, understanding the heterogeneous gene expression and molecular features of these subtypes is crucial. De-regulation of microRNA expression and activity has been shown to play an important role in tumor initiation and progression, including gliomagenesis. We have previously reported that expression of microRNA-128 (miR-128) is significantly down regulated in GBM and it diminishes self-renewal of GBM stem-like cells (GSCs) and sensitizes them to irradiation. Proneural-to-mesenchymal transition (PMT) manifested by concomitant up regulation of MES markers and down regulation of PN markers, is associated with increased malignancy, therapy-resistance and worse prognosis, but the underlying causes of PMT have not been convincingly characterized yet. In this study, we have demonstrated that miR-128 can regulate the PMT in GSC subsets. We showed that the expression of miR-128 in PN GSCs was significantly higher compared to MES subtype. As a tumor suppressive microRNA, miR-128 inhibited the MES GSC-specific high expression of Bmi1 and Suz12, two components of Polycomb Repressor Complexes (PRC) 1 and 2, respectively. In both GSC subtypes, miR-128 driven targeting of PRCs suppressed their epigenetic activity measured by ubiquitination of H2AK119 and tri-methylation of H3K27. Stable down regulation of miR-128 in PN GSCs significantly increased the expression of MES-specific gene signature (BCL2A1, CD44, WT-1, LYN, and MET) while its stable up regulation in MES GSCs resulted in the restoration of PN specific gene signature (CD133, SOX2, NES, OLIG2, and NOTCH1). We also showed that stable expression of miR-128 in GSCs could regulate the process of irradiation-induced PMT. Our in vivo studies showed the anti-tumorigenic role of miR-128 in both PN and MES GSC-derived intracranial tumor models. Taken together, we demonstrated that altering levels of miR-128 was sufficient to cause or reverse PMT, most likely by targeting the level/functions of PRCs and their target genes in GBM.

Citation Format: Arun K. Rooj, Marco Mineo, Franz Ricklefs, Agnieszka Bronisz, Ennio Chiocca, Jakub Godlewski. The novel role of microRNA-128 in proneural to mesenchymal subtype transition in glioblastoma stem cells by targeting components of pro-oncogenic Polycomb Repressor Complex. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1929.
Abstract 1932: The importance of RNA editing enzymes in glioblastoma
Authors: Gallo Angela
Published: July 2016

This study was supported by AIRC.

Glioblastoma (or grade IV astrocytomas) is a highly heterogeneous and deadly malignant brain tumor and one of the most incurable form of cancer in human that urgently needs new therapeutic targets. There is great interest in elucidating all the molecular basis and functional importance of genetics and epigenetic mechanisms occurring in glioblastoma. In our laboratory, we investigate whether essential post-transcriptional event (such as the A-to-I RNA editing) is a possible molecular mechanism involved in the appearance/progression of glioblastoma.

A-to-I RNA editing modifies the nucleotide sequence of RNA target molecules. The ADARs (Adenosine Deaminases that Act on RNA) are the enzymes responsible for this conversion acting on pre-mRNAs or non-coding RNAs (such as microRNA). Inosine, is recognized by the translation and splicing machineries, as Guanosine. Therefore, ADARs can play a direct and critical role in generating alternative protein isoforms (by codon changes and splicing pattern alterations) and modulating gene expression levels (by editing microRNAs and/or 3’-UTRs). In mammals, there are three different ADAR enzymes (ADAR1-3). The deregulation of ADAR2 activity has been connected with several neurological human diseases and Adar2 knockout animals show a lethal neurological phenotype. We demonstrated a direct correlation between a progressive decrease of ADAR2 editing activity and the increased grade of malignancy in paediatric astrocytomas (from grade I to grade IV). We show that the rescue of ADAR2 in glioblastoma cells is able to significantly inhibit in vivo glioblastoma tumor growth. Differently, when we decrease the expression of ADAR2 in malignant but not tumorigenic astrocytoma cell line (A172) we observed a boost of several cancers cell features such as cell proliferation, migration and colony formation.

By integrating deep-sequencing and array approaches with bioinformatics analyses and molecular studies, we observed that ADAR2 is essential to edit a small number of mature miRNAs and to significantly modulate the expression of about 90 miRNAs in glioblastoma cells that may contribute to cancer progression.

Our findings disclose an additional layer of complexity in miRNome regulation and provide information to better dissect the impact of ADAR2 editing enzyme in glioblastoma.

Citation Format: Angela Gallo. The importance of RNA editing enzymes in glioblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1932.
Abstract 1935: A role for microRNAs in the regulation of transcription factor ATF5
Authors: Gaither Kari A. , Liu David X. , Madarampalli Bhanupriya
Published: July 2016

ATF5 is a widely expressed transcription factor that modulates survival, proliferation, and differentiation and plays a role in homeostasis and cellular stress response. In unstressed conditions, ATF5 has a short half-life and is rapidly degraded due to post-translational modifications. Conversely, ATF5 is upregulated in cells under cellular stress. Furthermore, we have found that ATF5 is elevated in transformed C6 glioma and MCF7 breast cancer compared to nontransformed cells and is a survival factor for both transformed cell lines. Although it is known that ATF5 mRNA can be stabilized at the 5’ untranslated region (UTR) and protected from degradation via interactions with other proteins, regulation of ATF5 expression is not fully understood. We hypothesize that microRNA (miRNA) play a role in regulating the expression of ATF5 at the 3’ UTR. MiRNAs are endogenous small non-coding RNAs 20-25 nucleotides in length that contribute to regulation of gene expression at the translational level. Herein, we sought to identify the presence of specific miRNA and their levels and ability to downregulate ATF5 during cellular stress and other physiological conditions in vitro. To date, no studies have examined the regulation of ATF5 by miRNA. Binding sites of miRNA are typically found in the UTRs of target mRNAs. We used TargetScan, miRanda, and other in silico modeling programs to identify miRNAs that were predicted to bind to the 3’ UTR of ATF5 and selected miRNAs 129-5p, 433-3p, and 520b as initial candidates for their potential in regulation of ATF5. Here, we have employed multiple strategies to investigate a potential role of miRNAs in the regulation of ATF5. Luciferase reporter assays were used as an initial method of validation for the miRNA candidates. Subsequently, transfections were carried out with precursor microRNA and expression levels of ATF5 were measured under varying physiological conditions via Western blot analysis. Additionally, immunoprecipitations were carried out with ATF5 3’ UTR mRNA and HeLa cell lysate to assess the presence of miRNAs and quantify the amount of specific miRNA bound to the 3’ UTR under varying physiological conditions via qPCR analysis. Preliminary data show that miRNA are bound to the 3’ UTR of ATF5 and that miRNAs 129-5p, 433-3p, and 520b help to regulate the expression of ATF5 under varying stress conditions and at steady state. Better understanding of the regulation of ATF5 could have implications in a broad range of human malignancies, including glioma and potentially several other types of cancer.

Citation Format: Kari A. Gaither, David X. Liu, Bhanupriya Madarampalli. A role for microRNAs in the regulation of transcription factor ATF5. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1935.
Abstract 1949: MicroRNA-137 is a putative predictive biomarker and can inhibit cell proliferation and induce apoptosis in colorectal cancer
Authors: HUANG. YU-CHUAN
Published: July 2016

MicroRNA-137 (miR-137) is a potential tumor suppressor in many kinds of cancers, including colorectal cancer (CRC). However, the role of miR-137 in CRC remains unclear. Our study showed that miR-137 is silenced in human colorectal cancer tissues and colon polyps. The methylation of miR-137 promoter is determined by methylation-specific PCR (MSP) and quantified by pyrosequencing analysis. By bioinformatics analysis and Q-PCR assay, we confirmed that miR-137 can target COX2, CDC42, CDK6, and Aurora-A. The negative expression of miR-137 and Aurora-A is confirmed in human colorectal cancer tissues and colon polyps, as well as colon polyps from the AOM/DSS-treated mouse model. Enforcing expression of miR-137 in colorectal cancer cells resulted in G2/M arrest, reduced cell proliferation, and finally led to apoptosis. Those effects can be rescued by overexpressed Aurora-A. By xenograft animal model, the growth of tumors was attenuated in miR-137-overexpressing colorectal cancer cells, in which the tissues show an increased Annexin V signal. Additionally, the AOM/DSS-induced colorectal adenocarcinoma can be restored by demethylation drug treatment. Taken together, our study suggests that epigenetic silence of miR-137 may contribute to early CRC formation through inhibiting the expression of Aurora-A, and loss of miR-137 expression in colon polyps may serve as a potential biomarker to predict the tendency towards to CRC formation. The investigation of the regulatory mechanism of miR-137 in CRC progression may shed new light on early prognosis or cancer therapy for CRC in the future.

Citation Format: YU-CHUAN HUANG. MicroRNA-137 is a putative predictive biomarker and can inhibit cell proliferation and induce apoptosis in colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1949.
Abstract 195: Defining the role of PAK7 variants in melanoma
Authors: LaPak Kyle M. , Gross Michael A. , Vroom Denny C. , Lesinski Greg B. , Carson William E. , Burd Christin E.
Published: July 2016

Melanoma mortality is directly linked to its profoundly metastatic nature and inherent resistance to conventional chemotherapy. For this reason, increased understanding of the molecular mechanisms driving melanoma progression is of utmost importance. The p21-activated serine/threonine kinases (PAKs) are frequently mutated or overexpressed in cancer and have recently been implicated in tumor metastasis, proliferation, and apoptotic resistance. This observation has inspired drug discovery efforts to limit PAK activity; yet, mechanistic understanding of how PAKs (especially Type II PAKs) contribute to tumor initiation and progression is still evolving. PAK7, a type II PAK, is mutated in 16-18% of all melanomas. Melanoma-associated PAK7 mutations occur throughout the gene and have unknown biochemical and physiological consequences. The objective of this study was to determine how tumor-associated PAK7 variants mechanistically contribute to melanoma initiation and progression. First, we characterized PAK7 protein expression in a panel of 39 melanoma cell lines and primary melanocyte cultures. PAK7 levels were similar in both tumorigenic and non-tumorigenic cells and failed to correlate with the status of common melanoma driver mutations (e.g. NRASQ61, BRAFV600). In an array of 101 human tumor biopsies, PAK7 expression did not significantly change with advancing melanoma stage; however, PAK7 levels were higher in regional lymph node biopsies when compared to primary skin lesions. Since mutation is the predominant mechanism affecting PAK7 in melanoma, we investigated the biochemical and physiological consequences of common PAK7 variants. 11 distinct PAK7 mutations were stably expressed at low levels in VMM39 (melanoma) and NCI-H441 (lung) cells. Using these clones, changes in the phosphorylation of downstream PAK7 targets associated with cellular migration (p120), proliferation (CRAF), and apoptotic resistance (BAD, MDM2) were examined. In addition, we defined phenotypic alterations in cellular morphology, proliferation, migration, and apoptotic resistance associated with the expression of each mutant. These data allowed us to classify PAK7 variants of unknown significance into groups associated with specific cellular outcomes. To our knowledge, this is the first study to functionally characterize a wide variety of melanoma-associated PAK7 mutations and represents a critical first step in understanding how this kinase contributes to tumor initiation and progression.

Citation Format: Kyle M. LaPak, Michael A. Gross, Denny C. Vroom, Greg B. Lesinski, William E. Carson. Defining the role of PAK7 variants in melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 195.
Abstract 1958: Developing an online tool to validate survival-associated miRNAs utilizing expression data from 2,061 breast cancer patients
Authors: Győrffy Balázs , Lánczky András , Bottai Giulia , Nagy Ádám , Szabó András , Santarpia Libero
Published: July 2016

Background. MicroRNAs (miRNAs) are small non-coding RNAs capable of simultaneously regulating multiple gene networks, and affecting breast cancer patients’ outcome. Here we present the development of an online tool for the real-time meta-analysis of published miRNA datasets to identify novel prognostic biomarkers in breast cancer.

Methods. First, a comprehensive database was set up by searching GEO, TCGA, and PubMed repositories to identify datasets with published miRNA expression and clinical data. Due to platform differences, each dataset was processed separately. Kaplan-Meier and Cox regression analyses were performed to investigate the prognostic value of a set of dysregulated miRNAs in breast cancer. The complete analysis tool can be accessed online at: www.kmplot.com/mirna.

Results. Overall, 2,061 samples from four independent datasets were integrated into the tool including the expression signature of 1,302 distinct miRNAs. In addition to overall survival data, ER status (n = 2,039), histological grade (n = 1,396) and lymph node status (n = 1,933) are also available. By using our online tool, we demonstrated the prognostic value of some of the previous reported miRNAs implicated in breast cancer. For instance, miR-29c, miR-34c, miR-101, miR-125, and miR-190b, showed a consistent prognostic value in at least three out of four breast cancer datasets.

Discussion. In summary, we established the first online integrated platform for the rapid identification of the prognostic value of miRNAs in breast cancer.

Citation Format: Balázs Győrffy, András Lánczky, Giulia Bottai, Ádám Nagy, András Szabó, Libero Santarpia. Developing an online tool to validate survival-associated miRNAs utilizing expression data from 2,061 breast cancer patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1958.
Abstract 1961: Characterization of MicroRNA Expression during the Development of Resistance to a mTOR-Inhibitor in renal cell carcinoma
Authors: Heinzelmann Joana , Bauer Franziska , Unteregger Gerhard , Schendel Darja , Weiland Anne , Stöckle Michael , Junker Kerstin
Published: July 2016

Introduction:

Second line therapy of metastatic renal cell carcinoma (RCC) using the mTOR inhibitor RAD001 (everolimus) is hampered by the appearance of drug resistance. The cellular mechanisms leading to drug resistance are not yet clarified. Although the influence of miRNAs is proven for other systemic treatments, their impact on mTOR inhibitors is not investigated. Therefore, we analyzed miRNA alterations in correlation to development of resistance to RAD001 in RCC cell culture models.

Material and Methods:

The RCC cell line 786-O was treated with 1μM RAD001 for up to 20 passages after developing sunitinib resistance. TotalRNA was isolated at defined intervals. Resistance-development as well as drug efficacy during the treatment was monitored by proliferation assays. MiRNA Microarrays (Agilent Technologies) were performed using RAD001 sensitive and RAD001 resistant cells to identify differently expressed miRNAs. Results were validated by qRT PCR. Functional analyses are performed after transfection of RAD001 sensitive cells with miRNA candidates.

Results:

The Microarray analyses revealed a miRNA signature including 9 differently expressed miRNAs in RAD001 resistant cells compared to sensitive cells. QRT-PCR results showed that especially miR-302e was strongly up-regulated by the factor of four. At present, functional analyses of miR-302e are ongoing.

Conclusion:

To our knowledge this is the first study analyzing miRNA alterations during the development of resistance to mTOR inhibitors. We have shown that miR-302e expression is associated with RAD001 resistance in RCC cells. Since miR-302e is a known tumor suppressor modulating radio sensitivity and drug sensitivity in several tumors we are proving now if miR-302e can regulate the mTOR inhibitor resistance of RCC.

Citation Format: Joana Heinzelmann, Franziska Bauer, Gerhard Unteregger, Darja Schendel, Anne Weiland, Michael Stöckle, Kerstin Junker. Characterization of MicroRNA Expression during the Development of Resistance to a mTOR-Inhibitor in renal cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1961.
Abstract 1969: miRNA expression as potential biomarker for leiomyosarcoma and undifferentiated pleomorphic sarcoma
Authors: Pazzaglia Laura , Novello Chiara , Conti Amalia , Quattrini Irene , Pollino Serena , Picci Piero , Benassi Maria Serena
Published: July 2016

Rare and highly aggressive adult soft tissue sarcomas (STS), leiomyosarcoma (LMS) and undifferentiated pleomorphic sarcoma (UPS), have complex genomics characterized by a multitude of rearrangements, amplifications and deletions. Our previous data (Guled et al., Genes Chromosomes Cancer, 2014) showed 21 common differentially expressed miRNAs in UPS and LMS samples when compared to mesenchymal stem cells. On the base of these results, we performed target analysis by using bioinformatic tools (Diana-microt v3.0, Targetscan Vert release 5.2 and Pictar-Vert) and we identified 12 miRNAs potentially implicated in tumor development and progression.

The expression of the selected miRNAs was evaluated by TaqMan microRNA Array in 59 high grade STS primary samples (27 LMS and 32 UPS) stored at Rizzoli biobank. Ten tissues from non oncologic patients were used as control. Our results showed a significant lower expression of miR-1, miR-133b and miR-152 (respectively p = 0.0045, p = 0.0027 and p = 0.0016) in tumors when compared to nontumor tissue. Three different target prediction databases (miRBase, TargetScan, miRanda) recognized MET as common target gene of miR-1, mir-133b and miR-152 and KIT as target of miR-152. MET and KIT gene expression was validated in our series of STS by RT-PCR by using 2−ΔΔCT comparative method. The data demonstrated higher mean expression levels in STS than in control (respectively 2.08 and 0.15 for KIT; 18527.9 and 2324.9 for MET) without significant differences between two histotypes.

Our preliminary results confirmed MET and KIT as miRNA targets. Correlation with clinical data and in vitro studies with miRNA precursor transfected cell lines are on-going to better investigate their role as potential prognostic and therapeutic markers.

Citation Format: Laura Pazzaglia, Chiara Novello, Amalia Conti, Irene Quattrini, Serena Pollino, Piero Picci, Maria Serena Benassi. miRNA expression as potential biomarker for leiomyosarcoma and undifferentiated pleomorphic sarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1969.
Abstract 1972: Changes in the architecture, interactions and chromatin remodeling of SWI/SNF due to loss of Snf5
Authors: Bartholomew Blaine , Persinger Jim , Sen Payel , Dechassa Lemma Mekonnen , Luo Jie , Ranish Jeff
Published: July 2016

The mammalian SWI/SNF complex is a family of an estimated ∼100 complexes, each with different combinations of related subunits. Efforts in sequencing many cancer genomes has revealed that mammalian SWI/SNF complexes is one of the most frequently mutated epigenetic factors found in a broad range of cancers. In order to understand more about the subunit organization of SWI/SNF and because yeast and mammalian SWI/SNF complexes share many conserved subunits and domains; we have studied the role of the Snf5 subunit in the yeast SWI/SNF to better understand the role of the mammalian homolog SNF5 that is a known tumor suppressor gene. The basis for the loss of mammalian SNF5 being a driver mutation in pediatric rhabdoid tumors is currently not well understood or how the loss of SNF5 effects the composition, recruitment and nucleosome remodeling activity of the SWI/SNF complex. We have assessed the effects on deletion of Snf5 on the compositional integrity of SWI/SNF and have evidence for Snf5 forming a sub-complex with two other highly conserved subunits of SWI/SNF that are lost from the complex when Snf5 is deleted. In addition we have mapped the inter-subunit and intra-subunit interactions of the yeast SWI/SNF complex with lysine specific homo bi-functional crosslinkers and mass spectrometry. These data confirm the formation of a tri-subunit Snf5 sub-complex and show that this module uniquely interacts with the ATPase domain of the catalytic subunit of SWI/SNF (Snf2). These observations lead to a further investigation into the regulation of the remodeling activity of SWI/SNF by the Snf5 subunit. We have found that Snf5 is required for the ATPase domain to make stable interactions with nucleosomal DNA. And the loss of these interactions in turn also causes a reduction in the remodeling efficiency of the SWI/SNF complex. We have done additional mapping of the interactions of Snf5 as part of the SWI/SNF complex and find that the highly conserved region of yeast Snf5 corresponding to the human SNF5 protein associates with the surface of the nucleosome near the histone H2A-H2B dimer. We will discuss the relevance of these findings in highlighting how an aberrant form of SWI/SNF could be created by loss of Snf5 and provide an example of how it alters the normal function and structure of SWI/SNF.

Citation Format: Blaine Bartholomew, Jim Persinger, Payel Sen, Mekonnen Dechassa Lemma, Jie Luo, Jeff Ranish. Changes in the architecture, interactions and chromatin remodeling of SWI/SNF due to loss of Snf5. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1972.
Abstract 1987: Recurrent chromosomal translocations mediate reprogramming of Myb dependent regulatory networks in adenoid cystic carcinoma
Authors: Brayer Kathryn , Ness Scott A.
Published: July 2016

Adenoid Cystic Carcinoma (ACC), the second most common malignancy of salivary glands, is a rare tumor with bleak prognosis for which therapeutic targets are unavailable. Recently, we used RNA-sequencing (RNA-Seq) to analyze low-quality RNA from archival, formaldehyde-fixed, paraffin-embedded samples (1). In addition to detecting the most common ACC translocation, t(6;9) fusing the MYB proto-oncogene to NFIB, we also detected previously unknown t(8;9) and t(8;14) translocations fusing the MYBL1 gene to the NFIB and RAD51B genes, respectively. Mutated versions of c-Myb are potent oncoproteins, and Myb proteins are over- or highly-expressed in a wide variety of human tumors. Interestingly, ACC tumors with MYB and MYBL1 translocations displayed similar gene expression profiles, and the combined MYB and MYBL1 expression correlated with outcome, suggesting that the related Myb proteins are interchangeable oncogenic drivers in ACC.

While RNA-Seq provides information about gene fusions, alternative RNA splicing and gene expression signatures, this type of data does not address how translocations alter transcription factor function. Previous studies in our lab, and others, have demonstrated that Myb proteins contain C-terminal regulatory domains and are known to interact with several proteins via C-terminal motifs (2). These regulatory and interaction sites are lost upon chromosomal translocation in ACC; however, the downstream affect of this loss is not understood. Do the translocations merely relieve Myb transcription factors of repressive regulatory signals, thereby globally increasing Myb-dependent gene expression or, does the altered protein allow novel protein-protein interactions which re-target Myb transcription factors? Furthermore, the most frequent translocations involve the relocation of a large enhancer regions. What role does the recruitment of novel enhancer regions play in up-regulating Myb protein expression? Using a combination of ChIP-Seq and digital genomic footprinting, we are attempting to understand the mechanism of regulatory rewiring observed in ACC tumors.

References

1. Brayer et al. Recurrent Fusions in MYB and MYBL1 Define a Common, Transcription Factor-Driven Oncogenic Pathway in Salivary Gland Adenoid Cystic Carcinoma. Cancer Discovery. In press.

2. George and Ness (2014) Situational Awareness: Regulation of the Myb Transcription Factor in Differentiation, the Cell Cycle and Oncogenesis. Cancers: 6, 2049-2071.

Citation Format: Kathryn Brayer, Scott A. Ness. Recurrent chromosomal translocations mediate reprogramming of Myb dependent regulatory networks in adenoid cystic carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1987.
Abstract 1989: Multiple functional implications of MYB in ovarian cancer
Authors: Srivastava Sanjeev Kumar , Singh Seema , Bhardwaj Arun , Carter James E. , Rocconi Rodney P. , Scalici Jennifer , Singh Ajay P.
Published: July 2016

Ovarian cancer (OC) is the most common and deadliest gynecologic malignancies in the United States, with an estimated 21,290 new cases and 14,180 deaths in 2015. Therefore, it is highly imperative to identify novel molecular targets driving OC progression, so that an effective treatment strategy can be developed against this devastating disease. In the present study, we investigated the role of MYB in OC pathogenesis. MYB/c-MYB is a cellular progenitor of v-MYB oncogenes, which encodes for a transcription factor, and confers its oncogenic activity by regulating the expression of several target genes. First, we examined the expression of MYB in clinical specimens and OC cell lines by immunohistochemical and immunoblot analysis, respectively. We observed intense staining of MYB in the tissues of all the subtypes of OC, while no or negligible staining was observed in normal ovarian tissues. Moreover, we found MYB to be expressed in all the OC cell lines examined, with its heightened expression in highly aggressive (TOV112D, SKOV3-ip) and chemo-resistant (A2780-Cip) OC cell lines. To examine the pathological significance of MYB in OC, we silenced its expression in TOV112D, SKOV3-ip and A2780-Cip OC cell lines. Functional studies demonstrate that silencing of MYB led to decrease in growth and clonogenic ability in TOV112D, SKOV3-ip and A2780-Cip cells, as compared to their scrambled sequence-transfected (Scr) control cells. Furthermore, MYB-silencing reduced motility and invasion, as well as resulted in the loss of mesenchymal and gain of epithelial markers. In addition, our data showed that TOV112D-shMYB, SKOV3-ip-shMYB and A2780-Cip-shMYB were more sensitive to cisplatin-cytotoxicity as compared to their respective controls. Altogether, our studies provide the first experimental evidence for a functional role of MYB in altered growth, malignant behavior and chemoresistance of ovarian cancer.

Citation Format: Sanjeev Kumar Srivastava, Seema Singh, Arun Bhardwaj, James E. Carter, Rodney P. Rocconi, Jennifer Scalici, Ajay P. Singh. Multiple functional implications of MYB in ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1989.