bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3072: Characterization of TCL1-Tg:p53-/- mice that resemble human chronic lymphocytic leukemia with 17p-deletion: alterations in p53→Mir30→EZH2 axis.
Authors: Liu Jinyun , Ogasawara Marcia A. , Chen Gang , Pelicano Helene , Huang Peng
Published: August 2014

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the United States and Europe. CLL patients with deletion of chromosome 17p, where the tumor suppressor p53 gene is located, often develop a more aggressive disease with poor clinical outcomes. However, the underlying mechanism remains unclear. We have recently generated mice with Eμ-Tcl1-Tg:p53-/- genotype and showed that these mice develop aggressive leukemia that resembles human CLL with 17p deletion. In the present study, we further demonstrated that the p53 deficiency in the Tcl1 transgenic mice resulted in significant down-regulation of microRNAs miR-15a and miR16-1, associated with a substantial up-regulation of Mcl-1, suggesting that the p53-miR15a/16-Mcl-1 axis may play an important role in CLL pathogenesis. Interestingly, we also found that loss of p53 resulted in a significant decrease in expression of the miR-30 family in leukemia lymphocytes from the Eμ-Tcl1-Tg:p53-/- mice. Consistently, primary leukemia cells from CLL patients with 17p deletion also showed a decrease in the miR-30d expression. To further exam the biological significance of decrease in the miR-30 family in CLL, we investigated the potential involvement of EZH2 (enhancer of zeste homolog 2), a component of the Polycomb repressive complex known to be a downstream target of miR-30d and plays a role in disease progression in several solid cancers. RT-PCR and western blot analyses showed that both EZH2 mRNA transcript and protein levels were significantly increased in the lymphocytes of Eμ-Tcl1-Tg:p53-/- mice relative to Eμ-Tcl1-Tg:p53+/+ mice. Exposure of leukemia cells isolated from Eμ-Tcl1-Tg:p53-/- mice to the EZH2 inhibitor 3-deazaneplanocin (DZNep) led to induction of apoptosis, suggesting EZH2 may play a role in promoting CLL cell survival and this may contribute to the aggressive phenotype of CLL with loss of p53. Our study reveals that the p53-miR30-EZH2 axis may play an important role in CLL pathogenesis, and EZH2 may potentially be a target for CLL treatment.

Citation Format: Jinyun Liu, Marcia A. Ogasawara, Gang Chen, Helene Pelicano, Peng Huang. Characterization of TCL1-Tg:p53-/- mice that resemble human chronic lymphocytic leukemia with 17p-deletion: alterations in p53→Mir30→EZH2 axis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3072. doi:10.1158/1538-7445.AM2013-3072
Abstract 308: δ-Catenin/NPRAP/Neurojungin mutation promotes prostate tumorigenesis in mice overexpressing Myc oncogene .
Authors: Nopparat Jongdee , Zhang Jiao , Chen Yan-Hua , Lu Qun
Published: August 2014

Prostate cancer is the most common non-cutaneous malignant neoplasm in men in Western countries. In humans, prostate cancer progresses from precursor lesions, termed prostatic intraepithelial neoplasia (PIN), to overt carcinoma confined to the prostate, and finally to metastatic disease that often results in lethality. Previous studies in our laboratory have shown that δ-catenin/NPRAP/Neurojungin (gene designation: CTNND2), a primarily neural-enriched protein in the brain of healthy individuals, is overexpressed corresponding to human prostate cancer progression. This finding has led us to investigate the roles of δ-catenin in prostate cancer progression. We utilized a novel transgenic model of mouse prostate cancer with overexpression of human Myc oncogene driven by rat-probasin promoter as the initiating event, which has been proven to mimic human disease conditions. These Myc overexpressing mice were further crossbred with δ-catenin mutant (δ-cat−/−) mice whose exon 9 of δ-catenin gene was disrupted. In this study, we examined the histological differences of mouse prostate in wild type and Myc overexpression with δ-catenin mutant mice (Myc/δ-cat−/−). We observed that morphological alterations characteristic of high grade PIN developed in luminal epithelial cells of transgenic mice by 6 weeks. These changes include an increase of nuclear size, multi-layering, and cribriform formation. Surprisingly, Myc/δ-cat−/- mice at 6 months of age displayed the dramatically increased tumor size and progression from PIN to adenocarcinomas. We also discovered that Myc/δ-cat−/- mice increased in both number of proliferating cells and cells undergoing apoptosis compared with that of wild type animals. However, in Myc/δ-cat−/- mice, proliferative index was greater than apoptotic index by 3.7 fold. Collectively, our findings suggest that mutation of δ-catenin may contribute to tumorigenesis of mouse prostate by enhancing the effects of Myc oncogene resulting in an increase of cell proliferation and thereby promoting prostate tumor growth. Supported by NIHCA111891 and NIHCA165202.

Citation Format: Jongdee Nopparat, Jiao Zhang, Yan-Hua Chen, Qun Lu. δ-Catenin/NPRAP/Neurojungin mutation promotes prostate tumorigenesis in mice overexpressing Myc oncogene . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 308. doi:10.1158/1538-7445.AM2013-308
Abstract 3080: DBP-RB, a novel RNA-binding protein promotes microRNA processing in the DNA damage response.
Authors: Han Cecil , Lu Xiongbin
Published: August 2014

Defects in the DNA damage response (DDR) and global dysregulation of microRNA (miRNAs) are considered hallmarks of many types of human cancer. Recent studies show that DNA damage such as irradiation, UV and genotoxic agents induce global changes in miRNA expression profile in a variety of cell types. DDR-regulated miRNAs are known to be involved in the initiation and progression of tumorigenesis and also modulate the sensitivity of cells to DNA damaging agents. However, mechanism about how miRNA expression is regulated in response to DNA damage stress is largely unknown. DBP-RB, one of RNA binding protein is known to be overexpressed or amplified in several types of human cancer including neuroblastoma, breast and colon cancer and the biological function of DBP-RB in the cancer is not determined yet. Here, we found that that DBP-RB is interacted with not only Drosha/DGCR8 but also Dicer microprocessors. We also found that DBP-RB is colocalized with DNA damage foci after DNA damage and associated with ATM (Ataxia Telangiectasia Mutated), RAD50 and NBS1 in the DNA damage signaling. DBP-RB knockdown results in increased resistant to genotoxic drugs and inhibition of cell proliferation. We investigated the level of 742 human miRNAs in the DBP-RB knockdown cells and compared with DNA damage induced miRNAs using miRNA quantitative PCR. DBP-RB knockdown cell showed significant changes in the expression levels of a subset of miRNA and in particular, 75% of DBP-RB dependent miRNAs were also regulated in DNA damage-dependent manner. RNA-Immunoprecipitation (IP) reveals that DBP-RB is associated with that specific subset of microRNAs and depletion of DBP-RB inhibits the function of Drosha microprocessor. Taken together, our findings suggest that DBP-RB function as a mediator by transmitting DNA damage signal to the miRNA processors to modulate miRNAs processing through binding Drosha/DGCR8 and Dicer.

Citation Format: Cecil Han, Xiongbin Lu. DBP-RB, a novel RNA-binding protein promotes microRNA processing in the DNA damage response. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3080. doi:10.1158/1538-7445.AM2013-3080

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 3081: miR-885-5p, a p53-activated microRNA, inhibits the Wnt/β-catenin signaling pathway and hepatocellular carcinoma metastasis.
Authors: Zhang Zhuhong , Yin Jing , Lv Dan , Liu Chenghu , Yang Jian , Chang Antao , Sun Peiqing , Chen Si , Stupack Dwayne , Tian Yaping , Xiang Rong
Published: August 2014

The regulation of hepatocelluar carcinoma (HCC) progression by microRNAs (miRNAs) has attracted attention in recent years. Our previous study reported that the levels of circulating miR-885-5p were significantly up-regulated in patients with HCC and chronic hepatitis B (CHB). However, the role of miR-885-5p in HCC metastasis and the underlying mechanisms remain unknown. In this study, we used in situ hybridization to analyze the expression of miR-885-5p in HCC tissues and found that its expression was negatively associated with TNM stage of HCC. Consistent with this, the expression of miR-885-5p was negatively correlated with the invasive and metastatic abilities of human HCC cell lines. Kaplan-Meier analysis revealed that miR-885-5p levels correlated with the survival of HCC patients. Furthermore, enforced expression of miR-885-5p in high-metastatic HCC cells inhibited migration and invasion in vitro and in vivo. In addition, we identified that miR-885-5p directly targeted genes encoding the β-catenin CTNNB1 and the transcription factor TCF4, leading to decreased activity of the Wnt/β-catenin signaling pathway, which in turn contributes to metastasis in various cancers, including HCC. Notably, our results also showed that p53 up-regulated the expression of miR-885-5p and significantly inactivated the Wnt/β-catenin signaling pathway in HCC cells. We found that miR-885-5p levels correlated with wild-type TP53 status in HCC tissues. Taken together, these findings suggest that miR-885-5p is a negative regulator of HCC metastasis and indicate a potent rationale for developing miR-885-5p as a potential therapeutic agent against HCC metastasis.

Citation Format: Zhuhong Zhang, Jing Yin, Dan Lv, Chenghu Liu, Jian Yang, Antao Chang, Peiqing Sun, Si Chen, Dwayne Stupack, Yaping Tian, Rong Xiang. miR-885-5p, a p53-activated microRNA, inhibits the Wnt/β-catenin signaling pathway and hepatocellular carcinoma metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3081. doi:10.1158/1538-7445.AM2013-3081

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 3082: miRNA-10b promotes medulloblastoma proliferation and survival via Bcl-2.
Authors: Pal Rekha , Greene Stephanie
Published: August 2014

Introduction: The role of microRNAs (miRNAs) in the pathology of medulloblastoma (MB), the most common malignant brain tumor in children, is being elucidated at present. miRNA expression profiling studies have revealed a number of miRNAs implicated in medulloblastoma initiation and progression by controlling the expression of various genes responsible for cell proliferation or cell death. The effect of miRNA-10b on MB growth and proliferation through transcriptional induction of Bcl-2, an anti-apoptotic cellular proto-oncogene and tumor promoter, was explored in this study.

Methods: Assays were carried out using six established MB cell lines and 13 primary MB samples. A screening cancer-specific miRNA array identified miRNA-10b as highly expressed in MB compared to normal cerebellum. Real-time PCR showed that miRNA-10b is significantly up-regulated (42-100 fold) compared to the normal cerebellum in all MB cell lines. Two MB cell lines (DAOY and UW228) were transiently transfected with control miRNA, miRNA-10b inhibitor, and miRNA-10b mimic for 48 to 72 hrs. The transfected cells were subjected to RT-PCR, MTT, apoptosis, clonogenic assay, and western blot analysis.

Results: Transfection of miRNA-10b inhibitor induced a significant down-regulation of miRNA-10b expression in the MB cell lines, while miRNA-10b mimic induced miRNA-10b expression as measured by RT-PCR. Down-regulation of miRNA-10b expression resulted in a significant down-regulation of mRNA and protein levels of Bcl-2 and Mcl-1, decreased proliferation (p ≤ 0.001) and induced apoptosis (p ≤ 0.001) as compared to control. Inhibition of miRNA-10B further blocked the colony-forming capability of MB cells (p ≤ 0.001). miRNA-10b mimic transfection exerted an opposite effect. Down-regulation of miRNA-10b resulted in abrogation of phosphorylation of Bcl-2 at Ser70, a site previously reported to be critical in inhibiting apoptosis. Analyses of miRNA-10b levels in 13 primary MB samples revealed that the 2 patients with high levels of miRNA-10b (more than 200-fold increase versus control) had multiple recurrences (4.5) and died within 5 years of diagnosis, compared with the 11 patients with low levels of miRNA-10b who had a mean of 1.2 recurrences and 40% long-term survival.

Conclusions: In conclusion, our data indicate that miRNA-10b may act as an oncomir in MB tumorigenesis, and reveal a previously unreported mechanism with Bcl-2 as an important mediator of miRNA-10b activity in MB cell survival. Targeting of miRNA-10b may provide a novel therapeutic approach in the treatment of MB.

Citation Format: Rekha Pal, Stephanie Greene. miRNA-10b promotes medulloblastoma proliferation and survival via Bcl-2. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3082. doi:10.1158/1538-7445.AM2013-3082
Abstract 309: Loss of IL-6 or Stat3 drives advanced prostate cancer by promoting p53 inactivation.
Authors: Pencik Jan
Published: August 2014

Prostate cancer is the second most common cause of death in men. Approximately 258000 men in worldwide die each year from prostate cancer. The Jak/Stat pathway (Jak - Janus kinase, Stat - signal transducer and activators of transcription) is an important signaling cascade in prostate cancer. Stat3 seems to play a fundamental role in the regulation of proliferation, apoptosis, angiogenesis and hormone-resistance in prostate cancer. Although there has been evidence for an oncogenic function of Stat3, observations from several laboratories suggest the opposite. Under certain conditions activation of Stat3 leads to decreased growth of prostate cancer cells in vitro (LNCaP) and can exert an inhibitory effect on prostate cancer xenografts. To elucidate the role of IL-6/Stat3 signaling in prostate cancer (PCa), we have established Pten-deficient (Ptenpc-/−) PCa mouse models with additional deletion of IL-6 or Stat3. Ablation of IL-6 in Ptenpc-/− mice led to increased tumor formation and signs of massive chronic inflammation. In addition, Ptenpc-/- IL-6−/- mice showed decreased expression of p53-dependent cellular senescence markers when compared to PCa in Ptenpc-/- mice. The concomitant Pten and Stat3 inactivation (Pten/Stat3pc-/−) enhanced cell proliferation and accelerated the prostate cancer progression as well as production of inflammatory cytokines. Pten/Stat3pc-/− mice showed also significant downregulation of crucial components of the p53-dependent cellular senescence pathway. Furthermore, Pten/Stat3pc-/- PCa mice revealed high expression of AR and P-Akt correlated with advanced prostate cancer. These data suggest that disruption of IL-6/Stat3 signaling promotes tumorigenesis in Ptenpc-/- induced prostate cancer via p53 inactivation.

Citation Format: Jan Pencik. Loss of IL-6 or Stat3 drives advanced prostate cancer by promoting p53 inactivation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 309. doi:10.1158/1538-7445.AM2013-309
Abstract 3093: AR responsive miR-148a targets p27 and regulates the cell cycle in LNCaP cells.
Authors: Lombard Alan , Libertini Steve , Mudryj Maria
Published: August 2014

Prostate cancer is a commonly diagnosed malignancy and represents the second leading cause of cancer-related death in US men. Prostate cancer is initially dependent on androgens for survival and androgen signaling is mediated by the androgen receptor (AR). Due to the androgen dependency of prostate cancer, castration-therapy is a common course of action to combat the progression of this disease. However, almost inevitably, androgen independent prostate cancer later arises for which few strongly effective treatment options exist. In many of these cases, despite the androgen refractive nature of the disease, AR signaling is still necessary for the survival of the cancer but the downstream mechanisms that allow the AR to influence cellular proliferation remain to be fully understood. Further research elucidating these pathways is needed to discover novel therapeutic options for this disease. It has been shown that the AR represses the function of the cyclin dependent kinase inhibitor p27 but the way in which this happens is not completely known and seems to be multi-pronged. Research from Murata et al. (2010) demonstrated that miR-148a is an androgen responsive microRNA. Furthermore, it was shown by Guo et al. (2011) that miR-148a targets p27 in gastric cancer. Here we attempt to link these lines of research and uncover a novel pathway in which the relationship between the AR and p27 is mediated by miR-148a. We validated previous results showing that inhibition of AR signaling leads to an increase in p27 expression at both the protein and mRNA levels in the androgen responsive prostate cancer cell line LNCaP. It was shown that AR inhibition leads to an ∼2 fold decrease in miR-148a expression. Using a microRNA mimic, we demonstrated the ability of miR-148a to target p27 expression at both the protein and mRNA levels. We next showed that the miR-148a mimic can rescue the attenuated androgen signaling phenotype by reducing p27 levels and further demonstrate that this treatment leads to positive regulation of the cell cycle. Interestingly, inhibition of function using an antagomir against miR-148a leads to attenuated growth in LNCaPs suggesting a putative therapeutic angle by targeting this pathway. These results demonstrate a novel pathway mediated by the AR in which a miRNA is expressed which potentiates the cell cycle by targeting p27.

Citation Format: Alan Lombard, Steve Libertini, Maria Mudryj. AR responsive miR-148a targets p27 and regulates the cell cycle in LNCaP cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3093. doi:10.1158/1538-7445.AM2013-3093

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 3094: MiRNAs 484 and 210 control Pax-5 expression and function in breast cancer.
Authors: Harquail Jason M. , Robichaud Gilles A.
Published: August 2014

Every hour, approximately 21 people are diagnosed with cancer where 9 will succumb to their disease. Recent studies have enabled the identification of important factors regulating cancer progression, one of these being the Pax-5 gene. Pax-5, an essential developmental factor, is aberrantly expressed in various B cell cancer lesions and solid tumors such as carcinoma. Although Pax-5 downstream activity is well characterized, the regulation of aberrant Pax-5 expression in a cancer specific context is poorly understood. To investigate the regulation of Pax-5 expression, we turned our attention to micro-RNAs (miRNAs). MiRNAs are highly conserved, small non-coding RNA molecules that regulate key biological processes. Extensive studies also show their deregulation in multiple cancer lesions. This study aims to elucidate a correlation between differentially expressed miRNAs in cancer cells and deregulated Pax-5 expression levels. Using bioinformatics platforms we observe that miRNAs 484, and 210 are aberrantly expressed in breast cancer cells and cross-reference with their capacity to target the Pax-5 mRNA 3’ untranslated region (UTR 3’). Using anti-miRNAs transfected into Pax-5 expressing breast cancer cell lines (MCF-7 and MB231), we demonstrate that miRNAs 484 and 210 are capable of regulating Pax-5 expression. In addition, miRNA-regulated Pax-5 expression resulted in a concomitant alteration in Pax-5-dependant phenotype and cancer processes. This is the first study demonstrating the regulation of Pax-5 expression and function by non-coding RNAs in cancer cells. We believe that the aberrant expression of Pax-5 in cancer cells is in part due to deregulated miRNA expression profiles. This study will bring insight in regards to cancer regulating processes associated with miRNA and Pax-5 deregulations, thus helping us to better understand the aberrant Pax-5 expression levels within cancerous states. This study can therefore provide the eventual possibility of earlier, more efficient diagnostics as well as more targeted treatments for cancer patients.

Citation Format: Jason M. Harquail, Gilles A. Robichaud. MiRNAs 484 and 210 control Pax-5 expression and function in breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3094. doi:10.1158/1538-7445.AM2013-3094
Abstract 3105: Orphan nuclear receptor NR4A2 exhibits oncogenic activity in lung cancer cells.
Authors: Cao Chunxia , Zhang Min , Gao Ruli , Cress Douglas , Chen Zirong , Wu Lizi , Kaye Maria Zajac , Kaye Frederic
Published: August 2014

LKB1 (also known as STK11) is a frequent target for mutations in lung cancer, however, the role of LKB1 loss in tumorigenesis is undefined. NR4A2, together with NR4A1, and NR4A3, constitute the nuclear receptor (NR) subfamily 4A (NR4A), which are orphan NRs lacking identified ligands. NR4A family has been implicated in cell cycle regulation, apoptosis, inflammation, metabolism and more recently in carcinogenesis. However, there are conflicting data for tumorigenesis as NR4A1 and NR4A3 act as tumor suppressors in leukemia mouse models, while NR4A2 appears to stimulate tumor cell growth in vitro. Our laboratory discovered that the CRTC1 gene was aberrantly activated in LKB1-null cancer cells, stimulating the transcription of cAMP/CREB targets including NR4A gene members.

The aim of this study is to examine NR4A2 as a potential oncogene and therapeutic target under the regulation of CRTC1 in LKB1 wildtype and null lung cancer cells. We demonstrated here that somatic loss of LKB1 in tumor cells was associated with underphosphorylation and nuclear localization of CRTC1, this resulted in upregulated expression and stronger transcriptional activity of NR4A2 in LKB1 mutant lung cancer cells and clinical primary lung cancer samples. We confirmed that forskolin, which can activate adenylate cyclase then catalyze the transformation of ATP to cAMP, could induce CRTC1 dephosphorylation resulting in enhanced NR4A2 expression. We identified that activation of the CRTC gene family induced NR4A2 expression, whereas knockdown of CRTCs decreased the expression of NR4A2. Furthermore, short hairpin RNA-mediated down-regulation of NR4A2, results in attenuated lung cancer cell proliferation and colony formation in vitro and decreased the tumor sizes in vivo in nude mice.

In summary, our data suggested a CRTC-mediated transcriptional regulatory mechanism for NR4A2 activity, and proposed an oncogenic role of NR4A2 in LKB1 null lung cancer cell lines.

Citation Format: Chunxia Cao, Min Zhang, Ruli Gao, Douglas Cress, Zirong Chen, Lizi Wu, Maria Zajac Kaye, Frederic Kaye. Orphan nuclear receptor NR4A2 exhibits oncogenic activity in lung cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3105. doi:10.1158/1538-7445.AM2013-3105
Abstract 3107: Runt-related transcription factor 1 (RUNX1) is involved in transcriptional repression by estrogen receptor and breast cancer cell proliferation.
Authors: Liu Ka , Smith Richard L. , Nguyen-Vu Trang , Candelaria Nicholes R. , Rogerson Colin M. , Johnson W. Evan , Cheung Edwin , Jankovic Boris R. , alawi Wail Ba , Zhang Jinsong , Bajic Vladimir B. , Lin Chin-Yo
Published: August 2014

In the majority of breast cancers, estrogen and its cellular receptor, estrogen receptor (ER), are involved in regulating tumor cell proliferation. ER is both a prognostic marker and a therapeutic target in the management of hormone-dependent tumors. Current research efforts and the paradigm of ER transcriptional regulatory functions in breast cancer have focused on the role of ER in activating target gene transcription. Microarray studies and whole-genome ER binding site mapping experiments indicate, however, that ER can alternatively directly repress the expression of a significant subset of target genes. We posit that transcriptional repression by ER likely involves interactions with other co-regulatory transcription factors found in the proximity of ER binding sites in the cis-regulatory regions of repressed target genes and have identified the runt-related transcription factor 1 (RUNX1), a key regulator of hematopoiesis and leukemogenesis, as a candidate co-regulatory factor involved in transcriptional repression by ER. Here, we demonstrate that RUNX1 interacts with ER, specifically the ligand-binding domain, and is involved in the repression of ER target genes and estrogen-dependent and -independent proliferation of breast cancer cells. We also show that putative ER-RUNX1 target genes are associated with disease outcome in breast cancer patients. These findings not only provide the first evidence for the involvement of RUNX1 in hormonal carcinogenesis and additional insights into mechanisms of transcriptional regulation by ER but may potentially lead to more effective strategies for targeting ER and estrogen signaling in the prevention and treatment of breast cancer.

Citation Format: Ka Liu, Richard L. Smith, Trang Nguyen-Vu, Nicholes R. Candelaria, Colin M. Rogerson, W. Evan Johnson, Edwin Cheung, Boris R. Jankovic, Wail Ba alawi, Jinsong Zhang, Vladimir B. Bajic, Chin-Yo Lin. Runt-related transcription factor 1 (RUNX1) is involved in transcriptional repression by estrogen receptor and breast cancer cell proliferation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3107. doi:10.1158/1538-7445.AM2013-3107
Abstract 3108: A novel role for hPygo2 in ribosomal RNA transcription.
Authors: Andrews Phillip G. P. , Kao Kenneth R.
Published: August 2014

Background: Several ribosomes are required for each translated messenger RNA. During cell growth and division, a significant proportion of cellular resources are devoted to the production of ribosomes, but the epigenetic mechanisms by which cells adjust to this requirement are not fully understood. We have previously demonstrated that human Pygopus 2 (hPygo2) is highly expressed in, and required for the growth of a number of different cancers. hPygo2 functions as a chromatin remodeling protein through direct association with trimethylated Histone H3 at lysine4 (H3K4me3). It is here that hPygo2 likely serves as an adapter to which histone acetyltransferases can bind and promote further chromatin remodeling events required for transcription. We now present evidence that hPygo2 is involved in chromatin remodeling at the ribosomal (r)RNA promoter during proliferative growth of cancer cells. Methods: Immunoprecipitation (IP) and immunofluorescence (IF) was performed in breast (MCF7), ovarian (SKOV3) and cervical (HeLa) cancer cells. siRNAs were designed to specifically deplete hPygo2. For hPygo2 binding to the ribosomal gene promoter, chromatin IPs were used and analyzed by conventional or quantitative (q) PCR. 47S rRNA expression was measured by qPCR or by pulse-chase with either Br-UTP or 3H-uridine. Cell cycle analysis was performed by flow cytometry. Results: We found that hPygo2 interacted with UBF-1 and Treacle, two nucleolar proteins involved in 47S rRNA transcription. hPygo2 co-localized with UBF-1 and Treacle along with newly synthesized de novo rRNA in the nucleoli of cancer cells. hPygo2 was further detected at the ribosomal gene promoter along with core components of the rDNA transcription complex, including RNA polymerase I. Furthermore, hPygo2 was required for Histone H4 acetylation at the rRNA promoter and for transcription of the 47S pre-rRNA. Depletion of hPygo2 was accompanied by activation of the ribosomal stress response, detected by the binding of ribosomal protein L11 to hdm2, thereby inhibiting its E3 ubiquitin ligase activity resulting in p53 dependent growth arrest. Conclusion: Our results suggest that chromatin remodeling function of hPygo2 may serve to augment or maintain 47S pre-rRNA transcription required for ribosome biogenesis in cancer.

Citation Format: Phillip G. P. Andrews, Kenneth R. Kao. A novel role for hPygo2 in ribosomal RNA transcription. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3108. doi:10.1158/1538-7445.AM2013-3108
Abstract 3111: Expression of chromobox homolog 7 (Cbx7) is associated with poor prognosis in ovarian clear cell adenocarcinoma via regulating TRAIL-induced apoptotic pathway.
Authors: Yamashita Yoriko , Shinjo Kanako , Yamamoto Eiko , Akatsuka Shinya , Takahashi Takashi , Kajiyama Hiroaki , Kikkawa Fumitaka , Toyokuni Shinya
Published: August 2014

Ovarian cancer is the most lethal gynecologic malignancy, and among them, clear cell adenocarcinoma of the ovary (OCCA) has relatively worse prognosis associated with chemoresistancy. Chromobox (Cbx) 7 is a component of the polycomb repressive complex 1 that prolongs the lifespan of normal human cells via down-regulating the expression of INK4a/ARF and thus related with cell-cycle progression. However, recent reports studying the relationship between Cbx7 expression and the patient's survival differs among the cell origins of the tumors, and the precise role of Cbx7 in human carcinomas remains obscure. In this study, we analyzed the expression of Cbx7 by immunohistochemistry in 81 OCCA patients and evaluated the association with their clinical outcome. Both overall survival and progression-free survival of the patients were significantly shorter in Cbx7-positive than Cbx7-negative (P<0.05) tumors in OCCA. Cbx7 knockdown experiments using two OCCA cell lines, TOV21G and KOC-7C, revealed that the viability of the cells were significantly reduced compared to control (P<0.001). Expression microarray analysis revealed that apoptosis-related genes, especially TRAIL, were significantly upregulated in Cbx7 knockdown cells (P<0.01). We further confirmed that Cbx-7 knockdown resulted in TRAIL-induced apoptosis in OCCA cells. Thus, in this study, we showed for the first time that Cbx7 is associated with worse prognosis in OCCA. We also successfully demonstrated that the TRAIL pathway is a novel target of Cbx7 in these cells, and therapeutic agents utilizing the TRAIL pathway may be specifically effective targeting OCCA.

Citation Format: Yoriko Yamashita, Kanako Shinjo, Eiko Yamamoto, Shinya Akatsuka, Takashi Takahashi, Hiroaki Kajiyama, Fumitaka Kikkawa, Shinya Toyokuni. Expression of chromobox homolog 7 (Cbx7) is associated with poor prognosis in ovarian clear cell adenocarcinoma via regulating TRAIL-induced apoptotic pathway. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3111. doi:10.1158/1538-7445.AM2013-3111

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 3114: Constitutively active signal transducer and activator of transcription-3: an oncogene that increases gap junctional intercellular communication.
Authors: Trotman-Grant Aaron , Geletu Mulu , Raptis Leda
Published: August 2014

Gap junctions are specialized intercellular channels that connect the cytoplasm of adjacent cells and mediate the direct exchange of small ions and molecules between them. Gap junctional intercellular communication (GJIC) is usually blocked, or downregulated, in cells transformed by oncogenes, such as Src. One of the Src effector pathways leading to transformation and GJIC suppression is the Ras/Raf/MEK (mitogen-activated protein kinase)/ERK (extracellular signal-regulated kinase) pathway that has a prominent etiological role in cell proliferation, differentiation and cell survival; inhibition of this pathway in vSrc transformed cells restores GJIC. In addition, the distinct downstream effector of Src that is obligatory for neoplasia is the signal transducer and activator of transcription-3 (Stat3). Stat3 has the intrinsic characteristic of being upregulated in a range of tumors, and a modified version, the constitutively active form of Stat3 (Stat3C), has been shown to function as an oncogene. Conversely, inhibition of Stat3 can suppress tumor growth. To examine the role of Stat3 upon the Src-mediated, GJIC suppression, Stat3 was downregulated in rat liver epithelial T51B cells expressing activated Src through treatment with the CPA7, Stat3 inhibitor, or through infection with a retroviral vector expressing a Stat3-specific shRNA. The extent of GJIC was determined by electroporating the fluorescent dye, Lucifer yellow, into cells grown on two co-planar electrodes of electrically conductive, optically transparent, indium-tin oxide, followed by observation of the migration of the dye to the adjacent, non-electroporated cells under fluorescence illumination. The results demonstrate that, contrary to inhibition of the Ras pathway, Stat3 inhibition in cells expressing activated Src does not restore GJIC. On the contrary, Stat3 inhibition in normal cells with high GJIC levels eliminated junctional permeability. Interestingly, our results also demonstrate that expression of Stat3C in T51B cells and human lung cancer SK-LuCi6 cells, which have extensive communication and low Src levels, increased GJIC. Therefore Stat3, although it is generally growth promoting and in an activated form can act as an oncogene, its function is actually required for and increases junctional permeability.

Citation Format: Aaron Trotman-Grant, Mulu Geletu, Leda Raptis. Constitutively active signal transducer and activator of transcription-3: an oncogene that increases gap junctional intercellular communication. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3114. doi:10.1158/1538-7445.AM2013-3114
Abstract 3121: c-Jun as a target in pancreatic cancer.
Authors: Sheahan Anjali V. , Khachigian Levon M.
Published: August 2014

Prognosis for pancreatic cancer is especially poor, with a 5% survival rate. Current FDA-approved therapeutics gemcitabine and erlotinib extend patient survival by a mere two months. As the disease is asymptomatic, patients are generally diagnosed at later stages of the disease. Consequentially, only 15-20% of patients present with resectable tumors. Further, aggressive metastasis often occurs after surgical intervention, resulting in the death of 75% of patients within five years. These statistics indicate an unmet need for more effective therapeutic strategies to combat pancreatic cancer.

c-Jun is a component of the AP-1 early response transcription factor. It is expressed in 87% of primary pancreatic cancer lesions and in several pancreatic cancer cell lines. c-Jun overexpression has been linked to metastasis in numerous cancer models and previous work in our laboratory has demonstrated the efficacy of a DNAzyme against c-Jun for the suppression of tumor growth and angiogenesis in in vivo models of basal and squamous cell carcinoma. We sought to explore the role of c-Jun in pancreatic cancer and the therapeutic potential of gene silencing of endogenous c-Jun.

We confirmed serum-inducible c-Jun expression at the mRNA level using qRT-PCR in two in vitro models of pancreatic adenocarcinoma, PANC-1 and MIA PaCa-2. This is consistent with Western blot data, which confirm expression of both c-Jun as well as its phosphorylated form (p-c-Jun) at the protein level. Optimal uptake of a FITC-labeled siRNA into these cell lines was achieved using DOTAP/DOPE transfection reagent. We subsequently demonstrated that the effective blockade of c-Jun and p-c-Jun could be achieved with siRNA targeting human endogenous c-Jun. At the functional level, while blockade of c-Jun had no apparent effect on cell proliferation, PANC-1 and MIA PaCa-2 cell migration and invasion were markedly reduced. This was demonstrated using transwell assays. Interestingly, this effect was enhanced in cells that were also treated with gemcitabine. Further, we showed that MMP-1 expression was inhibited in MIA PaCa-2 cells treated with siRNA targeting c-Jun. We have also identified Cystatin F, MDM2, MET, MMP-13 and NF2 as potential downstream targets of c-Jun using a human tumor metastasis PCR array.

This in vitro data confirms the expression and activation of c-Jun in pancreatic cancer cells, and demonstrate a functional role for c-Jun in pancreatic cancer cell migration and invasion, which underpin metastasis. We are in the process of validating these results in vivo employing shRNA to generate a stable knockdown of c-Jun in pancreatic cancer cells (MIA PaCa-2).

Citation Format: Anjali V. Sheahan, Levon M. Khachigian. c-Jun as a target in pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3121. doi:10.1158/1538-7445.AM2013-3121
Abstract 3123: The Notch pathway inhibits TGF-β signaling in breast cancer through HEYL-mediated crosstalk.
Authors: Liangfeng Han , Diehl Adam , Nguyen Nguyen K. , Korangath Preethi , Weiwen Teo , Zhang Zhe , Kominsky Scott , Argani Pedram , Landberg Goran , Sukumar Saraswati V.
Published: August 2014

Acquired resistance to Transforming growth factor-β (TGF-β) is a key step in the early stages of tumorigenesis. Mutations of TGF-β signaling components, often found in other cancers, are rare in breast cancer, and little is known about the development of this resistance in breast cancer. On the other hand, activation of Notch pathway is known to play a substantial role in promoting breast cancer development. We hypothesized that crosstalk between these two pathways occurs through HEYL, a basic helix-loop-helix (bHLH) transcription factor, and a known direct target of Notch. We found that HEYL is overexpressed in 40% of breast cancers. Expression of Notch increased HEYL expression while knockdown of RBP-J, a critical mediator of Notch, reduced HEYL expression in HS578T and MDAMB-231 breast cancer cells. Taking into account the contradictory biological effects of Notch and TGF-β signaling, we sought to examine whether HEYL inhibits the TGF-β pathway. TGF-β treatment or Smad3 overexpression significantly increased the luciferase activity of the TGF-β responsive reporter vector, p3TP-Luc, and the P15 (CDKN2B) gene promoter, but this transactivation was strongly inhibited by HEYL. Bimolecular fluorescence complementation assays confirmed the interaction between Smad 3 and HEYL; immunoprecipitation assays with deletion constructs showed that the Basic domain of HEYL interacts with the MH2 domain of Smad3, and that their interaction is necessary for HEYL to inhibit TGF-β signaling. In addition, using a tet-off inducible breast cancer cell model, HEYL was shown to transcriptionally up-regulate many genes that drive metastasis and tumor angiogenesis. Supporting this concept, HEYL/HER2 double transgenic mice showed nearly 50% increase in lung metastasis (20% vs 46%) compared to HER2/neu mice. Similarly, lung colonization occurred in 100% of mice injected with MDAMB231 while HEYL-shRNAs reduced incidence to 50%. Therefore, HEYL promotes breast cancer growth and metastasis in Smad-dependent and Smad-independent mechanisms.

Citation Format: Han Liangfeng, Adam Diehl, Nguyen K. Nguyen, Preethi Korangath, Teo Weiwen, Zhe Zhang, Scott Kominsky, Pedram Argani, Goran Landberg, Saraswati V. Sukumar. The Notch pathway inhibits TGF-β signaling in breast cancer through HEYL-mediated crosstalk. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3123. doi:10.1158/1538-7445.AM2013-3123
Abstract 3125: KAP1 promotes malignant properties of breast cancer cells and regulates the expression of multiple KRAB-ZNFs.
Authors: Addison Joseph B. , Galeeva Renata , Chen Dongquan , Pugacheva Elena N. , Ivanov Alexey V.
Published: August 2014

Background and Objective: Chromatin remodeling is fundamental for normal development and is universally affected in cancer cells. The largest family of human transcription factors, KRAB-ZNF repressors, and their essential cofactor KAP1 play important roles in chromatin organization and regulate multiple cellular processes such as cell differentiation, pluripotency, apoptosis, DNA repair and imprinting. Here, we explored the role of KAP1 and KRAB-ZNFs in breast cancer.

Methods: Using cancer patient samples, in vitro and animal xenograft model we analyzed KAP1 role in cancer cell proliferation and primary tumor formation.

Results: Patient sample analysis showed KAP1 and KRAB-ZNFs overexpression in significant proportion of breast tumors. KAP1 depletion in a panel of breast cancer cell lines inhibited cancer cell proliferation, while KAP1 overexpression stimulated cell growth. Concordantly, KAP1 knockdown in MDA-MB-231LN human breast cancer cells inhibited primary tumor growth in orthotopic xenograft mouse model, while KAP1 overexpression had an opposite, growth stimulatory effect. KAP1 depletion resulted in decreased expression of a number of cancer-associated genes. Functional in vitro studies provided evidence that KAP1 positively regulates expression of multiple KRAB-ZNFs.

Discussion and Conclusions: These results thus establish KAP1 as an oncogenic factor in breast cancer. Grant support: NIH NCRR grant P20 RR16440 and Susan G. Komen for the Cure grant KG110350.

Citation Format: Joseph B. Addison, Renata Galeeva, Dongquan Chen, Elena N. Pugacheva, Alexey V. Ivanov. KAP1 promotes malignant properties of breast cancer cells and regulates the expression of multiple KRAB-ZNFs. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3125. doi:10.1158/1538-7445.AM2013-3125
Abstract 3126: Insulinoma-associated 1 is a key transcriptional regulator of growth and survival-promoting pathways in small cell lung cancer.
Authors: Christensen Camilla L. , Shimamura Takeshi , Akbay Esra A. , Michaelsen Signe R. , Poulsen Hans S. , Wong Kwok-kin
Published: August 2014

Insulinoma-associated 1 (INSM1) is neuroendocrine (NE) transcription factor which temporal and spatial expression pattern is restricted to regions undergoing NE differentiation during embryogenesis. INSM1 is however re-expressed at high levels in small-cell lung cancer (SCLC) together with an array of other NE markers. The NE signature of SCLC is a central diagnostic tool for the disease but recent data pinpoints that this distinct signature might play a role in SCLC pathogenesis.

The functional role of INSM1 in SCLC was here investigated by shRNA-mediated silencing of INSM1 in a panel of SCLC cell lines. Upon silencing of INSM1, significant reduction in cell viability was observed as measured by MTT assay. This reduction in cell viability was concomitant with decreased cell proliferation and increased apoptosis as measured by BrdU incorporation and cleaved caspase 3/7 levels, respectively. Accordingly with phenotypic growth changes it was demonstrated that INSM1 silencing caused reduced phosphorylation of members of the growth- and survival-promoting PI3K/Akt, MAPK and JAK-STAT signaling pathways and reduced levels of the inhibitors of apoptosis proteins (IAPs) members c-IAP1, XIAP and survivin. Importantly, it was shown that INSM1 is an upstream regulator of signatures previously shown to play a role in SCLC tumorigenesis, including achate-scute homolog 1 (ASCL1) and Sonic Hedgehog (SHh) pathway. Upon silencing of INSM1, a decrease in mRNA and protein levels of ASCL1 and downstream targets aldehyde dehydrogenase 1 (ALDH1) and delta-like ligand 3 (DLL3) was observed. Furthermore, mRNA levels of the SHh positive mediators Gli2, Gli3 and Smoothened was downregulated while the SHh inhibitor Patched was upregulated upon INSM1 silencing. Futhermore, overexpression of the Notch1 intracellular domain in SCLC cells resulted in a significant reduction in INSM1 expression and cell viability. This suggest that a master inhibitor of the NE signature, Notch1 signaling, could be an inhibitor of INSM1-driven tumorigenesis.

In conclusion, INSM1 is a potential key driver of growth- and survival-promoting pathways in SCLC and warrants further investigation of INSM1 as a therapeutic target.

Citation Format: Camilla L. Christensen, Takeshi Shimamura, Esra A. Akbay, Signe R. Michaelsen, Hans S. Poulsen, Kwok-kin Wong. Insulinoma-associated 1 is a key transcriptional regulator of growth and survival-promoting pathways in small cell lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3126. doi:10.1158/1538-7445.AM2013-3126