bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2979: Wolf-Hirschhorn syndrome candidate 1 plays an important role in the pathogenesis of head and neck cancer.
Authors: Saloura Vassiliki , Cho Hyun-Soo , Lingen Mark , Li Terri , Gong Can , Tanguy Seiwert , Cohen Ezra , Hamamoto Ryuji , Nakamura Yusuke
Published: August 2014

Wolf-Hirschhorn syndrome candidate 1 (WHSC1) is a histone lysine methyltransferase (HMT) which was initially found to be deregulated in a subset of multiple myeloma cells with the t(4;14) (p16;q32) translocation and poor prognosis. Recently, our group as well as others found that WHSC1 was overexpressed in a variety of tumors, including lung and esophageal squamous cell carcinomas, with low or no expression in normal tissues except the testis. Long-term survival of patients with squamous cell carcinoma of the head and neck (SCCHN) remains poor and thus new therapeutic strategies are urgently needed. Epigenetic alterations have mainly been studied as a potential mechanism of progression from dysplasia to squamous cell carcinoma. Given the promising potential of HMTs as novel drug targets and the aforementioned interesting expression patterns, we further investigated the role of WHSC1 in carcinogenesis through immunohistochemical analysis (IHC) using tissue microarrays of 123 locoregionally advanced SCCHN patients, and 6 samples of fibroepithelial hyperplasia lesions obtained from oral biopsies. We classified its expression levels with a scoring scale of 0 (no expression), +1 (mild), +2 (moderate) or +3 (strong). WHSC1 expression in the nucleus and cytoplasm was moderate (score +2, 37%) or strong (score +3, 38%) in 75% of SCCHN samples, while 25% showed mild (score +1, 24%) or no (score 0, 1%) expression. WHSC1 staining of normal epithelial regions revealed an exclusively nuclear expression pattern in the basal and parabasal layers. This is consistent with reports suggesting a physiologic role of the Wnt-pathway, which was recently found to be induced by WHSC1, in the differentiation process of the squamous epithelium. We also performed Western blot analysis of WHSC1 in 8 SCCHN cells lines, 3 control fibroblast cell lines and 1 keratinocyte cell line. We observed moderate to strong overexpression of WHSC1 in 4 of 8 SCCHN cell lines, while no expression was detected in the normal keratinocytes or the fibroblast cell lines. These findings support a potential role of the WHSC1 pathway in the pathogenesis of SCCHN and provides a new therapeutic avenue for drug discovery in this fatal disease.

Citation Format: Vassiliki Saloura, Hyun-Soo Cho, Mark Lingen, Terri Li, Can Gong, Seiwert Tanguy, Ezra Cohen, Ryuji Hamamoto, Yusuke Nakamura. Wolf-Hirschhorn syndrome candidate 1 plays an important role in the pathogenesis of head and neck 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 2979. doi:10.1158/1538-7445.AM2013-2979
Abstract 2983: Gene resilencing following decitabine therapy is initiated by nucleosome reoccupancy and is related to CpG island shore methylation.
Authors: Patil Vibha , Sloane Mathew A. , Wong Jason Wh , Liu Jia , Ward Robyn L. , Hesson Luke B.
Published: August 2014

Decitabine is a front line therapy for myelogenous leukaemias and is in clinical trials for various solid tumour types. This DNA demethylating agent reactivates genes silenced by promoter hypermethylation in cancer. However upon drug withdrawal reexpressed genes undergo remethylation (resilencing) which may underlie drug resistance and presents a barrier to effective therapy. We aimed to define the ordered sequence of epigenetic events associated with resilencing. Using biallelically methylated MLH1 as a model gene, we profiled epigenetic changes at the MLH1 promoter associated with reexpression and resilencing in a colorectal cancer cell line before, during and after Decitabine treatment. In contrast to the closed chromatin structure observed before treatment, Decitabine induced increased MLH1 expression and 54% decreased promoter methylation. We show that gene resilencing, which occurs 6-8 days following removal of therapy, is not due to promoter remethylation but is initiated by reoccupancy of nucleosomes to demethylated promoter alleles. Furthermore, long-term monitoring of cells following treatment showed MLH1 expression never reverts to pretreatment levels with low-level expression and demethylated promoter alleles persisting up to 118 days after withdrawal of Decitabine. Genome-wide methylation profiling was used to categorise promoter CpG Islands (CGI) based on their degree of demethylation after treatment. This revealed that CGIs with persistence of demethylated promoter alleles after long-term recovery (n=108) showed significantly lower levels of CGI shore methylation (p=0.0231). Our findings suggest a role for shore methylation in susceptibility to CGI remethylation and that therapeutic targeting of nucleosome assembly may provide a novel strategy to prevent gene resilencing.

Citation Format: Vibha Patil, Mathew A. Sloane, Jason Wh Wong, Jia Liu, Robyn L. Ward, Luke B. Hesson. Gene resilencing following decitabine therapy is initiated by nucleosome reoccupancy and is related to CpG island shore methylation. [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 2983. doi:10.1158/1538-7445.AM2013-2983
Abstract 2997: SOX2 contributes towards invasive phenotype of serous ovarian cancer by up-regulating MMP2.
Authors: Mohapatra Gayatry , Kikuchi Junko , Birrer Michael J.
Published: August 2014

SOX2 encodes a member of the SRY-related HMG-box (SOX) family of transcription factors involved in the regulation of embryonic development and in the determination of cell fate. SOX2 maps to 3q26.33, a region frequently amplified in HGSOvCa as reported in the TCGA and other large array-based CGH studies. However, the role of SOX2 in HGSOvCa oncogenesis and progression has not been studied yet. Given the importance of SOX2 in maintenance of stem cell phenotype and its overexpression in solid tumors, we sought to define the role of SOX2 in HGSOvCa.

In order to establish a direct role for SOX2 in vivo, we conducted DNA copy number analysis and qRT-PCR analysis in two independent panels of primary HGSOvCa and 25 serous OVCA lines. We overexpressed SOX2 in T29, OVCA433, A2780, SKOV3 and OVCAR5 cells and/or knockdown SOX2 in UCI101 and OV90 cells. Ectopic expression of SOX2 resulted in decreased cell proliferation, soft agar colony formation, in vivo tumorigenicity but higher degree of invasion relative to cells with empty vectors. SOX2 overexpression increased MMP2 activity as determined by gelatin zymography. We also performed mRNA profiling to identify SOX2 target genes. SOX2 knockdown cells decreased migration,andinvasion but increased tumorigenicity. Differentially expressed genes in cells overexpressing SOX2 will be presented. Increased SOX2 expression in HGSOvCa appears to produce a more invasive yet quiestant cell. Overexpression of SOX2 may induce a stem-like transcriptional program in HGSOvCa. Thus, identifying SOX2 target genes may offer new insight into the understanding of ovarian cancer biology.

Citation Format: Gayatry Mohapatra, Junko Kikuchi, Michael J. Birrer. SOX2 contributes towards invasive phenotype of serous ovarian cancer by up-regulating MMP2. [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 2997. doi:10.1158/1538-7445.AM2013-2997
Abstract 3000: New epigenetic biomarker for colorectal cancer.
Authors: Takane Kiyoko , Midorikawa Yutaka , Yagi Koichi , Sakai Ayako , Aburatani Hiroyuki , Takayama Tadatoshi , Kaneda Atsuhi
Published: August 2014

DNA is released and circulate in the blood of cancer patients. Epigenetic changes in the levels of circulating DNA has been associated with tumor burden and malignant progression. Public physical examination recommends occult fecal blood test for colorectal cancer (CRC) screening but its false positive rate is too high. As for serum marker for CRC including CEA and CA19-9, their sensitivity are nor satisfactory especially in early stage CRC patients and therefore, the potential use of circulating DNA for CRC screening has emerged. The aim of this study is the clinical utility of cell-free DNA as blood biomarkers. Based on our previous report about the new methylation markers for CRC on a genome-wide scale, we selected 4 methylation markers positive for CRC and established CRC screening panel. First, we collected plasma DNA from blood samples of 100 CRC patients preoperatively and 50 age-matched healthy volunteers. Then, we performed pyrosequence about plasma DNA after bisulfite treatment. Using this cancer screening panel, 82 CRC patients were positive for at least 1 marker. On the other hand, no control samples were positive for any markers and therefore, sensitivity and specificity using this cancer screening panel for CRC were 82.0% and 100%, respectively. According to TNM classification, positive rate for earlier stage including 1 and 2 patients were lower than advanced CRC patients. Similarly, the patients with abnormal serum CEA level harbored higher positive rate for the cancer panel, but serum CA19-9 level which is another CRC marker was not associated with positive rate. Two markers of the panel were relatively high in old age although they were negative for control samples. Considering that no hypermethylation was detected in control samples, this cancer panel is superior to preexisting blood tumor markers for CRC and available for cancer screening.

Citation Format: Kiyoko Takane, Yutaka Midorikawa, Koichi Yagi, Ayako Sakai, Hiroyuki Aburatani, Tadatoshi Takayama, Atsuhi Kaneda. New epigenetic biomarker for colorectal 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 3000. doi:10.1158/1538-7445.AM2013-3000
Abstract 3013: Elucidating the functional role of microfibril-associated glycoprotein-2 (MAGP-2) in human ovarian cancer.
Authors: Na Young-Jeong , Vathipadiekal Vinod , Wei Wei , Kwok Tim , Birrer Michael
Published: August 2014

Epithelial ovarian cancer, is the most fatal gynecologic cancer, resulting in approximately 15,000 deaths annually in the US. Our understanding of molecular events relating to the patient survival and overall prognosis is not clear. Using transcriptional profiling technology combined with Cox regression analysis in microdissected serous ovarian cancers, we have identified microfibril-associated glycoprotein 2 (MAGP-2) as a strong prognostic factor toward poor patient outcome. Ectopic overexpression of MAGP-2 was employed to evaluate its functional role in ovarian cancer. MAGP-2 overexpression increased cell migration and resistance to cisplatin in vitro. Moreover, the conditioned medium from MAGP-2 overexpression cells promoted endothelial cell motility. In an orthotopic model using luciferase labeled ovarian cancer cells, MAGP-2 overexpression increased the intraperitoneal tumor growth. In addition, the tumor with high MAGP-2 expression showed increased microvessel density. Our results suggest MAGP2 positively modulates the ovarian cancer tumorigenecity and angiogenesis, which may subsequently lead to poor patient survival.

Citation Format: Young-Jeong Na, Vinod Vathipadiekal, Wei Wei, Tim Kwok, Michael Birrer. Elucidating the functional role of microfibril-associated glycoprotein-2 (MAGP-2) in human ovarian 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 3013. doi:10.1158/1538-7445.AM2013-3013
Abstract 3025: Receptor tyrosine kinase ErbB2/HER2 translocates into mitochondria and regulates cellular metabolism.
Authors: Ding Yan , Liu Zixing , Desai Shruti , Pannell Lewis , Yi Hong , Wright Elizabath , Owen Laurie , Dean-Colomb Windy , Fodstad Oystein , Lu Jianrong , Tan Ming
Published: August 2014

It is well accepted that ErbB2, an important oncoprotein, localizes on the plasma membrane as a receptor tyrosine kinase. This study describes a novel observation that ErbB2 localizes in mitochondria of multiple cancer cell lines and patient samples.

We identified an endogenous mitochondrial targeting sequence in ErbB2 and found that ErbB2 translocates into mitochondria through the association with mtHSP70, a key player in the canonical mitochondrial protein importation pathway. Additionally, we observed that mitochondrial ErbB2 (mtErbB2) negatively regulates mitochondrial respiratory functions. Oxygen consumption and activities of complex I, II and IV of the mitochondrial electron transport chain were decreased in mtErbB2-overexpressing cells. Mitochondrial membrane potential and the cellular ATP level also were decreased by mtErbB2. In contrast, mtErbB2 enhanced cellular glycolysis. The translocation of ErbB2 and its impact on mitochondrial function are kinase dependent. Mitochondrial ErbB2 regulates the phosphorylation and activity of COX II and consequently the cytochrome c release and apoptosis. Additional studies showed that cancer cells with higher levels of mtErbB2 were more resistant to Trastuzumab and deletion of the mitochondrial targeting sequence of ErbB2 sensitized the cells to Trastuzumab.

Our study provides a novel perspective on the metabolic regulatory functions of ErbB2 and reveals that mtErbB2 plays an important role in the regulation of cellular metabolism and cancer cell resistance to therapeutics. Since ErbB2 plays important roles in multiple organs and multiple types of cancers, the study may have broad impact on the fields of cancer biology.

The work was supported by The Vincent F. Kilborn Jr. Cancer Research Foundation and National Institutes of Health Grant RO1CA149646.

Citation Format: Yan Ding, Zixing Liu, Shruti Desai, Lewis Pannell, Hong Yi, Elizabath Wright, Laurie Owen, Windy Dean-Colomb, Oystein Fodstad, Jianrong Lu, Ming Tan. Receptor tyrosine kinase ErbB2/HER2 translocates into mitochondria and regulates cellular metabolism. [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 3025. doi:10.1158/1538-7445.AM2013-3025
Abstract 3027: A novel function of EGFR in epigenetic modulation via hMOF in breast cancer .
Authors: Lim Seung-Oe , Li Chia-Wei , Chang Shih-Shin , Shen Jia , Hung Mien-Chie
Published: August 2014

The epidermal growth factor receptor (EGFR) is aberrantly active in breast cancer, initiates and drives tumorigenic pathways, and has been implicated in multiple tumorigenic processes including hyperplasia, chemo-resistance and metastasis. Histone lysine modifications directly influence activation and repression of transcription. MYST family acetyltransferase, human males absent on the first (hMOF), plays an important role in transcription activity through histone H4K16 acetylation. Loss of hMOF and subsequently the absence of H4K16 acetylation have been shown to be a common feature in human cancer including breast cancer. However, the regulation of hMOF remains largely unknown. Recently, we have discovered the acetylation of EGFR in human breast cancer cells. Thus, we screened a set of EGFR-interacting acetyltransferases using co-immunoprecipitation analysis and ingenuity pathway analysis (IPA). Here, we found that EGFR directly interacts with and phosphorylates hMOF. EGFR-induced phosphorylation hMOF was significantly reduced in the hMOF YF mutant. Indeed, hMOF YF mutant showed a higher histone acetyltransferase (HAT) activity compare to wild type hMOF. In addition, tyrosine kinase inhibitor (TKI) also enhanced the HAT activity of wild type hMOF. Furthermore, we found that EGFR modulates acetylation status of histone H4K16 via phosphorylating hMOF. We anticipate our study of EGFR and hMOF to be of therapeutic relevance, possibly leading to novel discovery in drug development targeting the receptor tyrosine kinase and the acetylation process.

Citation Format: Seung-Oe Lim, Chia-Wei Li, Shih-Shin Chang, Jia Shen, Mien-Chie Hung. A novel function of EGFR in epigenetic modulation via hMOF 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 3027. doi:10.1158/1538-7445.AM2013-3027
Abstract 303: A tissue microarray-based study of the basal cell in canine prostatic hyperplastic, pre-neoplastic and neoplastic lesions.
Authors: Amorim Renée Laufer , Fonseca-Alves Carlos Eduardo , Rodrigues Marcela M p , Rogatto Silvia Regina
Published: August 2014

It has been stated that dog is the best natural model to study carcinogenesis in comparison with men. In addition, the loss of the basal cell layer integrity is one of the main characteristics in human pre-neoplastic and neoplastic prostatic lesions. In human normal prostate, the basal cells selectively express p63 in their nuclei, demonstrating basal cell layer integrity. However, in both primary and metastatic prostate carcinomas, has been described absence of p63 immunoexpression. This study aimed to evaluate the involvement of basal cells (p63 and 34βE12) and proliferative index (ki-67) in prostatic pre-neoplastic and neoplastic lesions from dogs. A tissue microarray (TMA) was constructed using 66 canine prostatic paraffin-embedded lesions, selecting appropriate and representative areas of normal tissue (n=7), benign prostatic hyperplasia (BPH, n= 18), prostatic inflammatory atrophy (PIA, n=22) and prostatic carcinoma (PCa, n=19). Immunohistochemistry was performed using peroxidase method and DAB. The immunohistochemical staining was assessed by scores according the distribution of positive cells for p63 and 34βE12 (score 1: <25% cells positive, 2: 26% to 50%, 3: being 51% and 75% and 4:> 75%) and for Ki67, positive nuclei were counted in each lesion. Chi-square or Fisher exact test was used to determine the association between the categorical variables and student t test to compare proliferative index among lesions. Concerning the p63 protein, we found higher number of cases with more than 75% of positive basal cells in CaP (p=0.002) and PIA (p= 0.0042) when compared to normal tissue and BPH. CaP and PIA showed 34βE12 protein expression when compared to normal prostatic tissue and BPH (p= 0.032, p=0.0064). There was no statistically significant difference in the comparison between PIA and BPH. Ki67 showed higher proliferative index in PCa (p= 0.0028) and PIA (p= 0.01) compared to normal tissue and BPH. Higher p63 and 34βE12 immunohistochemical staining as well as higher proliferative index in these cells were observed in canine pre-neoplastic lesion (PIA) and prostate carcinoma indicate the involvement of basal cells in these lesions in dogs, different from humans. These results do not support the dog as the best natural model to the study of all types of prostatic carcinomas in men.

Financial support: Fapesp

Citation Format: Renée Laufer Amorim, Carlos Eduardo Fonseca-Alves, Marcela M p Rodrigues, Silvia Regina Rogatto. A tissue microarray-based study of the basal cell in canine prostatic hyperplastic, pre-neoplastic and neoplastic lesions. [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 303. doi:10.1158/1538-7445.AM2013-303
Abstract 3038: Epithelial Membrane Protein-2 (EMP2) promotes glioblastoma growth via upregulating invasion and angiogenesis.
Authors: Qin Yu
Published: August 2014

Recent data suggests that EMP2 is a novel oncogene in a number of tumors including breast, ovarian, endometrial and primary central nervous system malignancies. Its expression correlates with poor survival and can serve as a prognostic indicator in many tumors. Glioblastoma multiforme (GBM) is an aggressive brain tumor with a medium survival of 18 months, and we have found that EMP2 is expressed in the majority of GBM tumors but not in healthy brains. In order to therapeutically exploit the expression of EMP2 on these tumors, we have developed recombinant antibodies to this oncogene. In experimental models, use of the anti-EMP2 antibodies showed profound efficacy in reducing GBM tumor load. We further dissected the underlying molecular mechanisms and found that EMP2 accelerated GBM tumor growth through increasing αvβ3 integrin surface expression in GBM cells, activating downstream FAK and Src kinases and promoting cell migration and invasion. Furthermore, EMP2 upregulated vascular endothelial growth factor (VEGF) in several GBM cell lines. This increase appeared to be functionally relevant as conditioned medium from GBM cells with forced overexpression of EMP2 increased human umbilical vein endothelial cell (HUVEC) migration and capillary tube formation, suggesting a role for EMP2 in the regulation of tumor angiogenesis. Our results suggest the importance of this oncogene in the control of the tumor microenvironment and that targeting EMP2 expression may be a novel therapy for GBM.

Citation Format: Yu Qin. Epithelial Membrane Protein-2 (EMP2) promotes glioblastoma growth via upregulating invasion and angiogenesis. [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 3038. doi:10.1158/1538-7445.AM2013-3038
Abstract 3048: The genome-wide contribution of microRNAs to gene regulation in Ewing sarcoma.
Authors: Herrero-Martin David , Kauer Maximilian , Emdal Kristina B. , Sienski Grzegorz , Fourtouna Argyrou , Kralovics Robert , Olsen Jesper V. , Kovar Heinrich
Published: August 2014

The identification of microRNA (miR) targets within a given cellular context still poses a largely unsolved problem. MiR targets are predicted based on the presence of seed sequences in the 3'UTR of mRNAs. Target candidate lists are filtered assuming mRNA destabilization, and validated by artificially forcing ectopic expression of miR analogs, or by interfering with miR expression by antagomirs. However, the expression level and amplitude of physiological miR variations is much smaller than experimentally induced miR perturbations. Crosslinking and immuno precipitation (CLIP) followed by sequencing of bound RNA allows monitoring of dynamic changes in miR/mRNA occupancy of RISC. However, it is unclear if increased miR activity will lead to increased target read counts due to elevated RISC occupancy or to decreased read counts as a consequence of target destabilization.

Ewing sarcoma (ES) expresses an oncogenic transcription factor, EWS/FLI1.To study the impact of miRs to the gene regulatory network downstream of EWS/FLI1, we used a combination of mRNA and miR expression profiling, ChIP-seq, PAR-CLIP of AGO2, AGO2 knockdown, and SILAC monitoring of proteome changes in an inducible EWS/FLI1 knockdown ES cell line. Our data suggest that EWS/FLI1 perturbs gene regulation primarily on the transcriptional level and miRs play only a modulatory role for gene expression. Furthermore EWS/FLI1 affects the expression of many miR biogenesis and miR activity as well as RISC associated proteins. Knockdown of EWS/FLI1 results in pronounced changes of miR specific PAR-CLIP spectra. Disabled RISC activity lead to only minor changes in genome-wide mRNA expression. Our results are consistent with a complex role of the miRNome in the fine tuning of gene regulatory networks in Ewing sarcoma.

Supported by grants EU-FP7 259348 and Austrian Science Fund FWF 24708-B21.

Citation Format: David Herrero-Martin, Maximilian Kauer, Kristina B. Emdal, Grzegorz Sienski, Argyrou Fourtouna, Robert Kralovics, Jesper V. Olsen, Heinrich Kovar. The genome-wide contribution of microRNAs to gene regulation in Ewing sarcoma. [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 3048. doi:10.1158/1538-7445.AM2013-3048
Abstract 3059: MicroRNA-140 dysfunction leads to hepatocarcinogenesis through NF-κB activation by impairing the regulation of NF-κB coactivator.
Authors: Takata Akemi , Otsuka Motoyuki , Yoshikawa Takeshi , Kishikawa Takahiro , Ohno Motoko , Yoshida Haruhiko , Koike Kazuhiko
Published: August 2014

Nuclear factor κB (NF-κB) is a transcriptional factor that regulates a set of genes that are critical to many cellular biological processes, including liver tumorigenesis. MicroRNAs (miRNAs) are small non-coding RNAs that regulate specific target gene expression.To determine the miRNAs which are involved in regulating NF-κB activation in the liver, we screened for 60 kinds of miRNAs expressing in hepatocytes that can modulate NF-κB activation. We identified that miRNA-140 suppressed NF-κB activation significantly.Because miRNA-140 dysfunction was frequently observed in human hepatocellular carcinomas, it was hypothesized that the impairment of miRNA-140 function may be involved in hepatocarcinogenesis through NF-κB activation. Nuclear receptor interacting protein 1 (NRIP1) which is coactivator of NF-κB was identified as a target of miRNA-140 and the increased NRIP1 in hepatocellular carcinoma cells this led to enhanced NF-κB activation and hepatocarcinogenesis. MiRNA-140 knockout mice were indeed prone to hepatocarcinogenesis and had a phenotype similar to results in vitro. These results indicate that miRNA-140 acts as a liver tumor suppressor and the impairment of miRNA-140 function may also lead to hepatocarcinogenesis.

Citation Format: Akemi Takata, Motoyuki Otsuka, Takeshi Yoshikawa, Takahiro Kishikawa, Motoko Ohno, Haruhiko Yoshida, Kazuhiko Koike. MicroRNA-140 dysfunction leads to hepatocarcinogenesis through NF-κB activation by impairing the regulation of NF-κB coactivator. [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 3059. doi:10.1158/1538-7445.AM2013-3059
Abstract 3070: Suppression of tumor cell invasiveness and in vivo tumor growth by microRNA-874 in non-small cell lung cancer.
Authors: Kesanakurti Divya , Maddirela Dilip , Chittivelu Subramanyam , Rao Jasti S. , Chetty Chandramu
Published: August 2014

MicroRNAs are a family of small non-coding RNAs that regulate expression of many genes involved in normal development as well as human disorders including cancer. Here, we show that miR-874 plays a tumor suppressor role in non-small cell lung cancer (NSCLC) in vitro and in vivo. Interestingly, in silico target prediction analysis revealed numerous pro-oncogenes associated with tumor initiation, angiogenesis, invasion and migration, including MMP-2, uPA, NFAT5, PAK3, PAK7, LIMD, PECAM1, VEGF-A, Stat3, CUX1 and FGF1 as the putative target genes of miR-874. Our preliminary in situ hybridization experiments demonstrated the diminution of miR-874 expression in lung cancer tissues as compared to its normal counterparts. Overexpression of miR-874 in CD133-positive NSCLC cancer stem cell (CSC) population led to a significant loss in CSC phenotype and enhanced sphere de-differentiation into epithelial-like morphology. Restoration of miR-874 expression drastically reduced the invasive ability of cells in comparison to mock and control-miR treated cells by suppressing the protein levels of MMP-2 and uPA. Further, miR-874 treatment decreased orthotopic tumor growth in nude mice as compared to mock and control-miR treatments. Further, in vivo orthotopic tumor experiments in nude mice revealed the significant decrease in the immunoreactivity of human anti-MMP-2 and anti-uPA in miR-874-treated tumor sections. In conclusion, our study highlights the possible tumor suppressor role of miR-874 in NSCLC-initiating cells and suggests miR-874 as a potential target in the future treatment of NSCLC.

Citation Format: Divya Kesanakurti, Dilip Maddirela, Subramanyam Chittivelu, Jasti S. Rao, Chandramu Chetty. Suppression of tumor cell invasiveness and in vivo tumor growth by microRNA-874 in non-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 3070. doi:10.1158/1538-7445.AM2013-3070

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 3079: Estrogen signaling promotes cancer stem cell expansion via microRNA-140 targeting of pluripotency-associated factor SOX2 in ERα positive breast cancer cells.
Authors: Zhang Yongshu , Eades Gabriel L. , Yao Yuan , Li Qinglin , Zhou Qun
Published: August 2014

Multiple reports indicate that the prospective tumor suppressor miR-140 is down regulated in breast tumors compared to normal breast tissues; however, the role of miR-140 in breast tumorigenesis remains unclear. We have identified a novel target for miR-140 in mammary epithelium, the core pluripotency transcription factor SOX2. It is well known that SOX2 regulates the self-renewal of embryonic stem cells, however, less is known concerning possible oncogenic roles of SOX2 in breast cancer. We found that miR-140 and SOX2 target mRNA exhibited inverse expression in breast tumors and normal breast tissues, which was also correlated with estrogen receptor alpha (ERα) status. We investigated the role of ERα signaling in the regulation of miR-140 and SOX2 in breast tumors and found that estrogen stimulation resulted in decreased miR-140 and increased SOX2 expression in mammary epithelium and breast cancer cells. We performed bioinformatics promoter analyses of the miR-140 promoter revealing several predicted estrogen response elements (ERE) in the miR-140 promoter. We examined estrogen-induced activation of these prospective EREs using luciferase reporters for a miR-140 promoter deletion series. Furthermore, we used chromatin-immunoprecipitation to reveal that ERα binds to an ERE flanking the miR-140 promoter, consequently suppressing miR-140 transcription. As estrogen signaling was previously implicated in the regulation of breast cancer stem cells (CSCs), we investigated whether the miR-140/SOX2 pathway might provide a mechanistic link between ERα signaling and CSC maintenance. We found that forced activation of the miR-140/SOX2 pathway decreased mammosphere formation (a surrogate measure of CSC self-renewal) and opposed estrogen-induced expansion of the well-characterized CD44(high)/CD24(low) CSC subpopulation in ERα+ breast cancer cells. These results indicate that an ERα/miR-140/SOX2 pathway critically regulates breast CSC maintenance in ERα+ breast tumors.

Citation Format: Yongshu Zhang, Gabriel L. Eades, Yuan Yao, Qinglin Li, Qun Zhou. Estrogen signaling promotes cancer stem cell expansion via microRNA-140 targeting of pluripotency-associated factor SOX2 in ERα positive breast 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 3079. doi:10.1158/1538-7445.AM2013-3079
Abstract 3083: A novel method of sonic hedgehog signaling activation in the chemoresistance of glioblastoma: A ligand-independent mechanism.
Authors: Munoz Jessian L. , Rameshwar Pranela
Published: August 2014

Glioblastoma Multiforme (GMB) is the most common and lethal adult primary tumor of the central nervous system. Despite current chemotherapy with concomitant surgical resection and radiotherapy, GBM 5-year survival is ∼3%. A better understanding of the cellular pathways in tumor proliferation and survival is required for the development of new treatments. Previously, the Sonic Hedgehog pathway has been shown to be dysregulated in chemoresistant GBM. Pharmacological inhibition of SHH signaling with Cyclopamine enhanced GBM chemosensitivity to temozolomide (TMZ). SHH receptor, PTCH1, is tonically repressed by the ligandbut is activated by by receptor-mediated endocytosis of SHH. Here we show SHH signaling in GBM is ligand-independent and is initiated by post-transcriptional regulation of PTCH1. MicroRNA are small non-coding RNA molecules known to regulate a number of cell processes such as development and oncogenesis. As far as we are aware there is no report on microRNA in the regulation of PTCH1. Knockdown of Dicer, a Type III RNAase, which is required for microRNA synthesis, increased the cellular toxicity to TMZ, thus revealing the importance of microRNA in GBM chemoresistance. We have indentified and characterized PTCH as a functional target of microRNA-9 (miR-9) in both non-neural cell lines as well as GBM cell lines. Overexpression of miR-9 in GBM cells results in downstream pathway activation and resistance to TMZ treatment. We also explored a number of mir-9/PTCH1-dependent mechanisms of chemoresistance and uncovered downstream upregulation of ATP-binding cassette drug efflux transporters, MDR1 and ABCG2. Taken together, our data indicate a role for miRNA9 in the acquisition of chemoresistance in GBMs and may serve as novel therapeutic targets.

Citation Format: Jessian L. Munoz, Pranela Rameshwar. A novel method of sonic hedgehog signaling activation in the chemoresistance of glioblastoma: A ligand-independent mechanism. [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 3083. doi:10.1158/1538-7445.AM2013-3083
Abstract 3097: microRNA-21 inhibitor potentiates anti-tumor effect of radiation therapy in vitro and in vivo.
Authors: Cho Bong Jun , Choi Eun Jung , Kim Hans H. , Lee David J. , Chun Sun Ha , Kim In-Ah
Published: August 2014

microRNA-21 (miR-21) plays significant roles in carcinogenesis and is highly expressed in diverse human cancers. In this study, we evaluated the potential of targeting miRNA-21 to overcome the radioresistance of human cancer cells having an activated PI3K-Akt signaling and also aimed to elucidate the mechanisms of radiosensitization, and the effect on epithelial- mesenchymal transition (EMT).

A panel of human cancer cells (U251, U87, A549, MDA-MB-468, SKBR-3 cells) were transfected with miR-21 led to up-regulation of PI3K-Akt signaling and increased radioresistance compared with the cancer cells that were treated with nonspecific control. In contrast, a specific inhibitor of miR-21, an antipeptide nucleic acid (PNA) attenuated PI3K-Akt signaling and radiosensitized a panel of human cancer cells we tested. Inhibition of miR-21 was associated with persistent γH2AX foci formation and down-regulation of p-DNA-PKcs. While ectopic overexpression of miR-21 exhibited higher invasion and migration potential and increased vascular tube formation, inhibitor of miR-21 decreased these typical features of EMT. Treatment of anti-miR-21 significantly decreased tumor burden in nude mice bearing intracranial U251 xenografts compared to control as measured by bioluminescence. Combined treatment of anti-miR-21 and radiation further decreased tumor burden compared to each treatment alone.

In summary, miR-21 is an important onco-miR which confers radioresistance and diverse features of EMT. Inhibition of miR-21 could be a potential strategy for improving the efficacy of radiation therapy via unique modulation of PI3K-Akt signaling and EMT.

Citation Format: Bong Jun Cho, Eun Jung Choi, Hans H. Kim, David J. Lee, Sun Ha Chun, In-Ah Kim. microRNA-21 inhibitor potentiates anti-tumor effect of radiation therapy in vitro and in vivo. [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 3097. doi:10.1158/1538-7445.AM2013-3097
Abstract 3098: MiRNA-145 is a master regulator of the hallmarks of thyroid cancer.
Authors: Boufraqech Myriem , Zhang Lisa , Jain Meenu , Gulati Neelam , Nilubol Naris , Kitano Mio , Xiong Yin , Kebebew Electron
Published: August 2014

microRNA-145 (miR-145) in an important member of the family of microRNAs which are downregulated in several types of tumors. However, miR-145 expression and function in thyroid cancer and in cellular differentiation is unknown. In our study, we performed miRNA profiling in normal, benign and malignant thyroid cancers including undifferentiated tumors with validation of differentially expressed miRNAs by quantitative RT PCR. We found significant downregulation of miR-145 in differentiated and undifferentiated thyroid cancer. We performed functional studies in thyroid cancer cell lines and found that miR-145 decreases cell proliferation, induces cell cycle arrest, decreases cell invasion and targets the PI3K/Akt pathway, one of the most commonly activated pathways in thyroid cancer and which is associated with epithelial-mesenchymal transition (EMT). Overexpression of miR-145 in cell lines also decreased the expression of EMT markers vimentin and N-cadherin. Furthermore, inhibition of miR-145 in normal primary thyroid cultured cells decreased NIS and PAX8 expression, key regulators of thyroid cell differentiation. miRNA-145 expression in tumor tissue was inversely correlated with VEGF serum levels in the same patients and overexpression of miR-145 in vitro decreased angiogenesis and VEGF secretion.

MiRNA-145 has important regulatory effects on the hallmarks of thyroid cancer cells and thus may be involved in thyroid cancer progression.

Citation Format: Myriem Boufraqech, Lisa Zhang, Meenu Jain, Neelam Gulati, Naris Nilubol, Mio Kitano, Yin Xiong, Electron Kebebew. MiRNA-145 is a master regulator of the hallmarks of thyroid 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 3098. doi:10.1158/1538-7445.AM2013-3098
Abstract 3103: Overexpression of homeoprotein six1 activates non-canonical TGF-beta signaling to induce epithelial-mesenchymal transition in human papillomavirus type 16-immortalized human keratinocytes.
Authors: Xu Hanwen , Pirisi Lucia , Creek Kim E.
Published: August 2014

Inappropriate expression of embryonic genes, particularly homeodomain transcription factors, contributes to tumorigenesis and tumor progression. The overexpression of Six1, a member of the Six family of homeodomain transcription factors, has been found in various human cancers, and is associated with increased tumor progression and metastasis. Previous studies in our laboratory discovered that the expression of Six1 mRNA and protein increased during in vitro progression of human papillomavirus type 16 (HPV16)-immortalized human keratinocytes (HKc/HPV16) toward a differentiation-resistant (HKc/DR) phenotype. However, the precise mechanisms of how Six1 promotes tumor onset at early and late stages of HPV16-mediated transformation remain unknown. In this study, we found that HKc/HPV16 and HKc/DR overexpressing Six1 exhibited a more mesenchymal phenotype compared to vector controls, as characterized by a fibroblastic morphology, and increased migration and invasion. We utilized Whole Human Genome Microarrays (4x44K from Agilent Technologies) to explore the gene expression changes associated with Six1 overexpression in HKc/HPV16 and HKc/DR. We found that overexpression of Six1 down-regulated epithelial-related genes (ex., E-cadherin and occludin) and up-regulated mesenchymal-related genes (ex., N-cadherin, vimentin, and snail), which strongly suggests that Six1 overexpression induces epithelial-mesenchymal transition (EMT). Gene expression changes determined by microarray analysis were confirmed by real time PCR and western blotting. Increased expression of TGF-beta receptors in response to overexpression of Six1 in HKc/DR did not lead to increased Smad-dependent signaling but did activate the Mitogen-activated protein kinase (MAPK) pathway, suggesting that MAPK non-Smad TGF-β signaling may be involved in Six1-mediated EMT. In conclusion, our study demonstrates that Six1 overexpression induces EMT, possibly by activation of the MAPK non-canonical (non-Smad) TGF-β pathway, during HPV16-mediated transformation of human keratinocytes.

Citation Format: Hanwen Xu, Lucia Pirisi, Kim E. Creek. Overexpression of homeoprotein six1 activates non-canonical TGF-beta signaling to induce epithelial-mesenchymal transition in human papillomavirus type 16-immortalized human keratinocytes. [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 3103. doi:10.1158/1538-7445.AM2013-3103
Abstract 3109: SOX4 and POU2F1 synergistically regulate metastasis-associated NRP1 in HCC metastasis.
Authors: Tsou Ann-Ping , Liao Yu-Lun , Sun Yi-Ming , Horng Jorng-Tzong , Hsiao Michael
Published: August 2014

Gene regulation by SOX proteins depends on the presence of requisite partners which provides target specificity and combinatorial control. Previously, we have shown that SOX4 is an oncogenic transcription factor involved in human hepatocarcinogenesis. By bioinformative prediction, we identified that POU2F1 is an SOX4 co-factor and further showed that SOX4 and POU2F1 synergistically regulate metastasis-associated NRP1 expression in HCC cells. Using co-immunoprecipitation experiments, SOX4 and POU2F1 were shown to participate in direct protein-protein interaction in vitro involving assembly into a complex that required the N-terminus and HMG domain of SOX4 interacting with POU2F1. In addition, RNAi knockdown of SOX4 expression of HCC cell lines drastically reduced the expression of epithelial-mesenchymal transition (EMT) related genes, N-cadherin, MMP2 and MMP9. SOX4 knockdown also reduced the expression of VEGF related genes VEGFC, FLT1 and FLT4 and blood vessel distribution in subcutaneous tumors was decreased in shSOX4, shPOU2F1 and shNRP1-infected Mahlavu tumor cells. Our results suggest that SOX4 and POU2F1 may play a critical role in the regulation of genes involved in tumor invasion. These results support a new regulatory network from SOX4 and POU2F1 to NRP1 that occurs during liver tumor metastasis. This investigation provides relevant gene targets that are regulated by SOX4 and POU2F1 in liver tumors and these results could be used to develop effective diagnostic and alternative therapeutic strategies for the treatment of intrahepatic HCC.

Keywords: SOX4; POU2F1; hepatocellular carcinoma

Citation Format: Ann-Ping Tsou, Yu-Lun Liao, Yi-Ming Sun, Jorng-Tzong Horng, Michael Hsiao. SOX4 and POU2F1 synergistically regulate metastasis-associated NRP1 in HCC 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 3109. doi:10.1158/1538-7445.AM2013-3109

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 3116: NF-κB2/p52 induces expression of inflammatory mediators in prostate cancer in vivo.
Authors: Nadiminty Nagalakshmi , Tummala Ramakumar , Lou Wei , Evans Christopher P. , Gao Allen C.
Published: August 2014

Introduction and objective: Inflammation plays a key role in the etiology of carcinoma of colon, cervix or liver, but PCa, in which inflammation has not been deemed to play a causative role, also display a pro-inflammatory gene profile. NF-κB is central to the induction of inflammation and the classical and alternative NF-κB activation pathways have been shown to modulate expression of inflammatory mediators. Here we explored the induction of markers of tumor-infiltrating inflammatory cells in two in vivo models overexpressing NF-κB2/p52.

Methods: LNCaP human PCa cells were infected with adenoviruses encoding empty vector or NF-κB2/p52 and injected s.c. into both flanks of nude mice. Gene expression of markers of inflammatory mediators such as macrophages, lymphocytes and dendritic cells was analyzed in the tumor tissues by qRT-PCR. In addition, whole prostates were collected from transgenic mice expressing NF-κB2/p52 in the prostate and RNA was analyzed for inflammatory markers. Expression of the cell surface markers was validated by immunohistochemistry in sections of whole prostates.

Results: The infiltration of host immune cells into the xenograft tissues was detected by expression of surface markers of cells of myeloid and lymphoid lineage. The expression of B220 (B cells), F4/80 (activated macrophages), Cd8A (CTL) was increased in p52-expressing xenografts compared to controls, indicating the presence of host-derived inflammatory cells in the xenografts. Expression levels of chemokines (Cxcl1), chemokine receptors (Ccr1, Ccr3) and pro-inflammatory cytokines (Il-6 and Tnf) were enhanced in the xenografts compared to the controls, indicating that expression of NF-κB2/p52 induces an inflammatory response in xenografts of human PCa cells.

Compared to littermate negative controls, expression of B220, F4/80, Cd8A and Cd208 was increased in transgenic mouse prostates expressing p52. Furthermore, expression levels of Cxcl1, Ccr1, Ccr3, Il-6 and Tnf were also enhanced compared to controls, indicating the induction of inflammation in mouse prostates by NF-κB2/p52.

Conclusions: Our previous studies have shown that NF-κB2/p52 plays an important role in the development of castration resistance in human PCa cells. In this study we showed that NF-κB2/p52 may induce an inflammatory response in xenografts of human PCa cells or in transgenic mouse prostates, and thereby promote tumor proliferation.

Citation Format: Nagalakshmi Nadiminty, Ramakumar Tummala, Wei Lou, Christopher P. Evans, Allen C. Gao. NF-κB2/p52 induces expression of inflammatory mediators in prostate cancer in vivo. [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 3116. doi:10.1158/1538-7445.AM2013-3116