bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5258: PTEN levels are controlled by a nuclear transport receptor in lung cancer.
Authors: Chen Muhan , Herzka Tali M. , Zeeman Martha E. , Plafker Kendra S. , Minderer Matthias , Castillo-Martin Mireia , Cordon-Cardo Carlos , Plafker Scott M. , Trotman Lloyd C.
Published: August 2014

The maintenance of PTEN protein levels is critical for tumor suppression. Yet, the ubiquitination system has been shown to affect PTEN levels both adversely through degradation, as well as positively through nuclear import, and it has remained unclear how these two processes are integrated to prevent cancer. Here we show, that a nuclear import receptor is at the heart of a failsafe system that maintains PTEN levels by mediating its nuclear transport. Loss of import receptor function not only leads to cytoplasmic PTEN accumulation but also prompts PTEN degradation through a novel component of the PTEN ubiquitination system.

By testing the consequences of importin loss in vivo, we found that hypomorphic mice developed lung adenocarcinoma, which presented with aberrant cytoplasmic PTEN localization and degradation, as predicted by our in vitro findings. Since the corresponding human locus suffers frequent deletion as well as inactivating mutations in lung cancer, we propose that this import receptor is a novel tumor suppressor that antagonizes PI 3-Kinase signaling in settings with at least one intact PTEN gene.

Citation Format: Muhan Chen, Tali M. Herzka, Martha E. Zeeman, Kendra S. Plafker, Matthias Minderer, Mireia Castillo-Martin, Carlos Cordon-Cardo, Scott M. Plafker, Lloyd C. Trotman. PTEN levels are controlled by a nuclear transport receptor in 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 5258. doi:10.1158/1538-7445.AM2013-5258
Abstract 5259: Ubiquitination and degradation of EGFR mutants by C-terminus hsp70 interacting protein (CHIP) in non-small cell lung cancer (NSCLC).
Authors: Lee Jeong Eun , Jang Kang Won , Cha Hye Rim , Kim Jo-ock , Jung Sung Soo , Park Dong Il , Park Myoung Rin , Chung Chaeuk
Published: August 2014

Several somatic mutations in the kinase domain of the epidermal growth factor receptor (EGFR), including L858R and exon 19 deletions, are predictive markers for EGFR tyrosine kinase inhibitor treating non-small cell lung cancer (NSCLC), especially adenocarcinoma. The mutational activation of EGFR is stabilized by Hsp90. Therefore Hsp90 inhibition may promote ubiquitin-mediated degradation of EGFR mutants. C-terminus hsp70 interacting protein (CHIP) has been shown to negatively regulate Hsp70 and Hsp90 function and to convert Hsp90 complexes from a chaperone function to one that promotes the ubiquitination and degradation of interacting proteins. Here, we report a novel interaction of EGFR mutants with an E3 ubiquitin ligase CHIP, which can ubiquitinate, and destabilize EGFR mutants. In CHO cells expressing either wild-type or mutant EGFR, inhibition of expression of its mutants: L858R, G719S and del L747_E749del, occurred following overexpression with CHIP, whereas diminution of wild-type EGFR was not in CHIP transfected cells. In full-down assay, CHIP especially interacted with G719S and del L747_E749del, but not L858R, and simultaneously induced ubiquitination of its proteins. Similarly, EGFR proteins in NSCLC cell lines harboring EGFR mutations, PC9, were also declined by CHIP mediated ubiquitination and degradation compared with the protein in wild-type cells. In EGFR mutant cell lines, CHIP inhitibited cell proliferation as well as depletion of phosphor-Akt and ERK. These data suggest that CHIP regulates and stabilizes EGFR mutants and that CHIP may be a novel regulator and marker in EGFR-mutants NSCLC. We confirmed these results by tumor xenograft model. Based on above findings, we conclude that CHIP is a new modulator in EGFR mutant pathway in NSCLC.

Citation Format: Jeong Eun Lee, Kang Won Jang, Hye Rim Cha, Jo-ock Kim, Sung Soo Jung, Dong Il Park, Myoung Rin Park, Chaeuk Chung. Ubiquitination and degradation of EGFR mutants by C-terminus hsp70 interacting protein (CHIP) in non-small cell lung cancer (NSCLC). [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 5259. doi:10.1158/1538-7445.AM2013-5259

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 5263: Src inhibition enhances BMP receptor antagonists downregulation of Id1 and growth suppression of lung cancer cells with a K-Ras mutation.
Authors: Langenfeld John E. , Langenfeld Elaine
Published: August 2014

The bone morphogenetic protein 2 (BMP2) is aberrantly expressed in 98% of non-small cell lung carcinomas. Our studies show that selective antagonists (DMH1, DMH2, and LDN) of the type I BMP receptors inhibit the proliferation and induce cell death of lung cancer cell lines, which is mediated at least in part by downregulating the expression of Id1 and Id3. The Id proteins are known to regulate essential aspects of tumorigenesis in many carcinomas.

The Src signaling pathway has been shown to increase the expression of Id1 in A549 cells. Src regulation of Id1 is mediated through the BMP pathway. Src binds to pSmad 1/5, which enhances its translocation into the nucleus. Src also mediates activity of response elements on the Id1 promoter that also bind pSmad 1/5. The BMP signaling pathway suppresses Src signaling, suggesting feedback inhibition.

We examined the ability of a BMP receptor antagonist (DMH2) and a Src antagonist (PP2) to regulate Id1 and Id3 expression in three lung cancer cell lines with a K-Ras mutation (A549,H157,H727) and 3 without a K-Ras mutation (H1299,H865,U1752). PP2 caused a reduction in Id1 and/or Id3 in 2 of the 3 cell lines with a K-Ras mutation (A549,H157). PP2 had no effect or increased the expression of Id1 and Id3 in cell lines without a K-Ras mutation. DMH2 decreased expression of Id1 and Id3 and caused growth suppression of all the cell lines. In the A549 cells, the combination of DMH2 and PP2 caused significantly greater reduction in Id1 promoter activity and growth inhibition than either agent alone. Inhibition of BMP signaling caused an increase in the expression of phosphorylated Src, suggesting feedback regulation.

The data suggests that Src inhibition leads to the downregulation of Id expression predominately in lung cancer cells with a K-Ras mutation. Inhibition of BMP signaling with receptor antagonists causes significant downregulation of Id1/Id3 and growth inhibition even in lung cancer cell lines with K-Ras mutations. Inhibition of BMP signaling may activate Src, suggesting that both BMP and Src antagonists are needed to suppress Id expression and inhibit tumor growth.

Citation Format: John E. Langenfeld, Elaine Langenfeld. Src inhibition enhances BMP receptor antagonists downregulation of Id1 and growth suppression of lung cancer cells with a K-Ras mutation. [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 5263. doi:10.1158/1538-7445.AM2013-5263
Abstract 5283: Regulation of tetraspanins CD151 and CD9 by micro-RNA in breast and prostate cancers.
Authors: Bond Danielle R. , Cairns Murray , Ashman Leonie K. , Weidenhofer Judith
Published: August 2014

Rationale: Tetraspanins are involved in modulating various cellular functions related to cancer progression, such as cell-cell adhesion, migration and signalling. The tetraspanin CD151 (pro-metastatic) is typically over-expressed in cancers, whereas CD9 (metastasis suppressor) is generally under-expressed in cancers. Increased CD151 protein expression in breast and prostate cancers significantly correlates with poor patient outcomes, whereas high CD9 protein expression in these cancers is associated with good patient prognosis. However, progress in developing CD151 and CD9 as prognostic biomarkers and therapeutic targets is hampered by a lack of understanding of the mechanisms controlling altered CD151 and CD9 protein expression in cancers.

Objective: miRNAs, which are small non-coding RNAs involved in post-transcriptional control, are known to be dysregulated in breast and prostate cancers. Bioinformatics analysis has identified around 250 miRNA, which are predicted to bind CD151 or CD9, thus we aim to investigate if miRNAs are able to regulate the expression of CD151 and CD9 in vitro using a range of breast and prostate cancer cell lines.

Methods and Results: Using a dual luciferase reporter assay the level of protein expression regulation via CD151 or CD9 3’UTR, where miRNA generally bind their targets, was determined in 13 breast and prostate cell lines. This analysis showed that the amount of targeting of CD151 and CD9 varied across these cell lines indicating a role for miRNA. Analysis of miRNA expression in normal and cancerous cell lines using miRNA expression arrays and qPCR has confirmed that some miRNAs which are predicted to target CD9 such as miR-25 are up-regulated in many prostate and breast cancer cell lines compared to non-tumourigenic cells. Conversely, miRNA were identified with different expression levels that are predicted to target CD151. To determine whether these miRNAs directly regulate CD151 or CD9, a dual luciferase 3’UTR reporter assay encompassing specific miRNA mimics is being used.

Conclusion: Altered miRNA expression is one way in which prostate and breast cancer cells might alter tetraspanin protein expression to allow cancer progression and metastasis.

Citation Format: Danielle R. Bond, Murray Cairns, Leonie K. Ashman, Judith Weidenhofer. Regulation of tetraspanins CD151 and CD9 by micro-RNA in breast and prostate cancers. [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 5283. doi:10.1158/1538-7445.AM2013-5283

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 5291: Non-coding satellite RNAs are highly expressed in cancer adjacent lesions of human pancreatic carcinoma and may promote oncogenesis .
Authors: Kishikawa Takahiro
Published: August 2014

It was believed that most satellite repeats in pericentric heterochromatin region were silenced and rarely transcribed into RNAs. However, a few reports suggest that non-coding satellite RNAs may play a significant role in cell division, heterochromatin remodeling or DNA damage such as other well-researched non-coding RNAs. In fact, a next generation digital gene expression method recently revealed that human satellite II (HSATII) transcripts are highly and specifically upregulated in human pancreatic tumors. To further explore these findings, we examined the precise localization of HSATII transcripts in human clinical pancreatic tissues by in situ hybridization using highly sensitive locked nucleic acid-modified oligonucleotide probes. Unexpectedly, HSATII RNAs were not detected in pancreatic ductal cancerous lesions, in almost all cases examined.

Instead, HSATII RNAs were clearly expressed in non-cancerous adjacent acinar cells, surrounding carcinoma tissues. On the other hand, none of normal or inflammatory pancreatic tissues derived from the ones without cancer expressed HSATII RNAs. While,

HSATII transcript expression in normal pancreatic ductal cells could not be induced in vitro even under stress conditions which mimic the tumor-adjacent environments, the cells with ectopic expression of HSATII transcripts showed the increase foci of gH2AX, which may reflect the increased genomic instability. These results suggest that the expression of HSATII may not be phenotypic changes in cancers but may directly contribute to the oncogenesis in the cells in precancerous lesions.

Citation Format: Takahiro Kishikawa. Non-coding satellite RNAs are highly expressed in cancer adjacent lesions of human pancreatic carcinoma and may promote oncogenesis . [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 5291. doi:10.1158/1538-7445.AM2013-5291
Abstract 5308: CD9, CD63 and CD82 tetraspanins expression profile in vulvar carcinomas.
Authors: Carvalho Katia C. , Rocha Rafael M. , Cunha Isabela W. , Soares Fernando A.
Published: August 2014

Background: Vulvar carcinomas are approximately 95 % of all vulvar tumors. This cancer presents good prognosis since early diagnosed, and survival depends of inguinal lymphonode commitment, with about 30% of patients presents lymphonode metastasis. The mechanisms involved in biological behavior of vulvar cancer remain unclear and its treatment involves physical mutilation and psychosocial factors of patients. Therefore, identification of features related with this tumors development or genesis can contribute to its knowledge and treatment. The tetraspanins are proteins that are involved in several biological processes including cell proliferation, adhesion, invasion and migration. Their reduction or lack of expression is frequently reported in metastatic and poor prognosis tumors. Our aim was to analyze CD9, CD63 and CD82 tetraspanins expression profile in vulvar carcinoma compared to normal tissue. Methods: All samples were obtained from Pathology Department of Hospital A. C. Camargo, Sao Paulo, Brazil. TMA block was constructed with 150 paraffin-embedded samples and was used in immunohistochemical assay. The tissue sections were incubated with specific antibodies against CD9, CD63 and CD82, following manufacturer's instruction. The slides were evaluated and scored semiquantitatively by assessing the staining intensity in tumor cells and the frequency of positive cells. Results: The Immunohistochemical analysis showed that all tetraspanins were expressed in tumor samples with different intensity and frequency. CD63 were expressed by a higher samples number (90%), with stronger staining both cytoplasm (79%) and nucleus (11%). Lack of CD63 expression was described in metastatic melanomas lesions, while no metastatic lesions shows higher levels of this protein. Seventy five percent of our samples expressed CD9, exclusively on cellular membrane. CD82 showed positive reaction in 45% of the samples with weakly or moderate staining, both nucleus and cytoplasm. The association of the downregulation of CD9 and/or CD82 with a worse prognosis has been shown in breast, cervical, endometrial, esophageal, head and neck, and others carcinomas. Moreover, CD82 is considered to be an important metastasis suppressor. Conclusion: Our preliminary results showed that tetraspanins may have an important role in vulvar carcinomas and CD82/CD63 nuclear expression must be better investigated. Further analyses are ongoing.

Citation Format: Katia C. Carvalho, Rafael M. Rocha, Isabela W. Cunha, Fernando A. Soares. CD9, CD63 and CD82 tetraspanins expression profile in vulvar carcinomas. [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 5308. doi:10.1158/1538-7445.AM2013-5308
Abstract 5320: miR-211 expression inhibits MMP-9-induced phenotype and tumor growth in lung adenocarcinoma cancer stem cells in vitro and in vivo.
Authors: Chetty Chandramu , Chittivelu Subramanyam , Rao Jasti S.
Published: August 2014

MicroRNAs are small non-coding RNAs that post-transcriptionally modulate gene expression by degrading or repressing the translation of target messenger RNAs (mRNA). MicroRNAs play fundamental roles in diverse biological and pathological processes, including cell proliferation, differentiation, apoptosis, and carcinogenesis. MicroRNAs have been revealed to be involved in various stages of cancer development, showing that abnormal miRNA expression plays a critical role in modulating expression of known oncogenes or tumor suppressor genes during cancer progression. In the present study, we observed that miR-211 expression was significantly lower in human non-small cell lung cancer (NSCLC) than their matched normal tissue samples, and we elucidated the tumor suppressor role of miR-211 using in vitro and in vivo models. We demonstrated that the expression of precursor of miR-211 (p-mir-211) inhibits radiation-induced invasive nature and tumor progression of NSCLC-initiating cells (NSCLC-ICs) in vitro and in vivo. Overexpression of miR-211 in NSCLC-ICs inhibited radiation-induced MMP-9 expression and invasive nature. Overexpression of miR-211 also inhibited epithelial to mesenchymal transition (EMT), which caused a decline in the mesenchymal markers vimentin, beta catenin and TGF-beta signaling molecules while increasing the epithelial marker, E-cadherin. Further, miR-211-treatment inhibited pre-established tumors in orthotopic mouse models. In addition, MMP-9 immunoreactivity decreased in tumor sections from mice that received miR-211 treatment as compared to tumor sections from mice that received control treatments. Finally, the apoptotic population was significantly increased in tumor sections from mice that received miR-211 treatment. Our data suggest miR-211 as a novel approach for the inhibition of radiation-induced invasion and tumor growth in NSCLC-ICs.

Citation Format: Chandramu Chetty, Subramanyam Chittivelu, Jasti S. Rao. miR-211 expression inhibits MMP-9-induced phenotype and tumor growth in lung adenocarcinoma cancer stem cells 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 5320. doi:10.1158/1538-7445.AM2013-5320
Abstract 5322: CD44+ prostate cancer cells are regulated by microRNA-708: A novel role of microRNA with potential prognostic and diagnostic significance in prostate cancer .
Authors: Saini Sharanjot , Arora Sumit , Majid Shahana , Shahryari Varahram , Yamamura Soichiro , Chang Inik , Chiyomaru Takeshi , Fukuhara Shinichiro , Deng Guoren , Tanaka Yuichiro , Dahiya Rajvir
Published: August 2014

A challenging aspect of prostate cancer (PCa) is the high rates of recurrence associated with the disease. About 40% of men with localized prostate cancer suffer from relapse after initial therapy. In these cases, androgen ablation therapy is used to reduce tumor burden/circulating PSA to low or undetectable limits. However, most patients suffer from disease recurrence, as monitored by rising PSA and tumor progression to hormone refractory/castration resistant prostate cancer that is essentially untreatable. Tumor recurrence has been attributed to the existence of tumor-initiating cells (TIC) within the bulk of the tumor; CD44 is an important marker for these cells. In this study, we demonstrate that miR-708 is a key negative regulator of CD44+ subpopulation of PCa cells. miR-708 was underexpressed in CD44+ cells from PCa xenografts. Reconstitution of miR-708 in PCa cell lines or CD44+ PCa cells led to decreased tumorigenicity in vitro. Intratumoral delivery of synthetic miR-708 oligonucleotides triggered regression of established tumors in a murine xenograft model of human prostate cancer. Conversely, miR-708 silencing in a purified CD44- population of PCa cells promoted tumor growth. Functional studies validated CD44 to be a direct target of miR-708. Also, we identified Androgen Receptor (AR) and the serine/threonine kinase AKT2, a member of the PI3K/AKT signaling pathway as functional targets of miR-708. Clinically, low miR-708 expression was associated significantly with poor survival outcome, tumor progression and recurrence in prostate cancer patients. In view of these results we propose that reduced miR-708 expression leads to prostate cancer initiation, progression and development by regulating expression of CD44, AR, AKT2, which in turn regulate proliferative, invasive and migratory abilities of prostate cancer cells. Therefore, miR-708 may represent a novel therapeutic target for prostate cancer.

Citation Format: Sharanjot Saini, Sumit Arora, Shahana Majid, Varahram Shahryari, Soichiro Yamamura, Inik Chang, Takeshi Chiyomaru, Shinichiro Fukuhara, Guoren Deng, Yuichiro Tanaka, Rajvir Dahiya. CD44+ prostate cancer cells are regulated by microRNA-708: A novel role of microRNA with potential prognostic and diagnostic significance in prostate 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 5322. doi:10.1158/1538-7445.AM2013-5322
Abstract 5344: IGF1-receptor expression is regulated by miR-100 in metastatic pancreatic cancer cells.
Authors: Huang Justin S. , Egger Michael , Grizzle William E. , McNally Lacey R.
Published: August 2014

Patients with pancreatic adenocarcinoma have the lowest 5-year survival and yearly rates of incidence are almost equivalent to the mortality rates. Long-term cure rates by standard therapies are disappointing due to disseminated disease at diagnosis and chemotherapeutic resistance. New therapeutic targets are necessary for decreasing the progression of pancreatic cancer and identifying targets specific to metastasis would improve patient care. In this study, we evaluated the levels of microRNA of metastatic and non-metastatic cell lines. The expression level of microRNAs and mRNAs were identified through microarray analysis by examining and comparing 5 pancreatic cancer cell lines, two that can metastasize in vivo (S2VP10, S2CP9) versus three that do not metastasize (MiaPaCa2, Panc-1, and ASPC-1). MicroRNA analysis indicated an increase in miR-100 and a decrease in miR-138 expression in metastatic cancer cells. Microarray analysis of differentially expressed mRNAs between metastatic and non-metastatic pancreatic cell lines also indicated increased insulin growth factor-1 receptor (IGF1-R) expression (p<0.0001) in metastatic pancreatic cancer cell lines compared to non-metastatic pancreatic cancer cell lines. To confirm these results from microarray analysis, western blot and immunocytochemistry was performed. Western blot revealed IGF1-R expression was 7 fold increased in metastatic cancer cell lines compared to non-metastastatic cell lines. In addition, downstream expressions of proteins GRB2 and phosphorylated PI3K also were increased in aggressive cancer cell lines. Immunocytochemistry confirmed the linkage of IGF1-R to miR-100 as cells transfected with miR-100 inhibitor showed a decrease in IGF1-R. However, cells transfected with a miR-138 mimic did not affect IGF1-R expression. IGF1-R may be an important molecular feature and a useful biomarker for diagnosis and treatment of metastatic pancreatic cancer.

Citation Format: Justin S. Huang, Michael Egger, William E. Grizzle, Lacey R. McNally. IGF1-receptor expression is regulated by miR-100 in metastatic pancreatic 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 5344. doi:10.1158/1538-7445.AM2013-5344
Abstract 5349: Aberrant trimethylation of histone H3 lysine 27 is induced by chronic inflammation in mouse colonic epithelial cells, and is involved in the formation of a field for cancerization.
Authors: Takeshima Hideyuki , Ikegami Daigo , Wakabayashi Mika , Niwa Tohru , Kim Young-Joon , Ushijima Toshikazu
Published: August 2014

A field for cancerization (field defect), where genetic and epigenetic alterations are accumulated in normal-appearing tissues, is involved in human carcinogenesis, especially cancers associated with chronic inflammation. While aberrant DNA methylation is involved in the field defect and induced by chronic inflammation [Ushijima and Hattori, Clin Cancer Res, 2012], it is still unclear for trimethylation of histone H3 lysine 27 (H3K27me3), which is involved in gene repression independent of DNA methylation and functions as a pre-mark for aberrant DNA methylation. In this study, using a mouse colitis model induced by dextran sulfate sodium (DSS), we aimed to clarify whether or not aberrant H3K27me3 is induced by inflammation and involved in a field defect.

Analysis of H3K27me3 levels in colonic epithelial cells by chromatin immunoprecipitation combined with CpG island microarray analysis (ChIP-on-chip analysis) revealed that H3K27me3 levels were increased or decreased for 266 genomic regions by aging, and more extensively (27 increased and 3,782 decreased regions) by colitis. Among the 3,809 regions with alterations by DSS-induced colitis, 212 regions overlapped with those whose alterations were induced by aging. Analysis of the temporal profiles of H3K27me3 levels in the course of DSS treatment revealed that increase or decrease of H3K27me3 was induced as early as two weeks after the initiation of DSS treatment, and persisted at least for 16 weeks, even after the inflammation disappeared. Some of the aberrant H3K27me3 in colonic epithelial cells was carried over into colon tumors. Furthermore, H3K27me3 acquired at Dapk1 by colitis was followed by increased DNA methylation, supporting its function as a pre-mark for aberrant DNA methylation [Takeshima et al., Carcinogenesis, in press]. Regarding mechanisms of aberrant H3K27me3 induction, expression changes of polycomb-related genes including Ezh2, Kdm6a, and Kdm6b, were not involved.

These results demonstrated that aberrant H3K27me3 can be induced by exposure to a specific environment, such as colitis, and suggested that aberrant histone modification, in addition to aberrant DNA methylation, is involved in the formation of a field defect.

Citation Format: Hideyuki Takeshima, Daigo Ikegami, Mika Wakabayashi, Tohru Niwa, Young-Joon Kim, Toshikazu Ushijima. Aberrant trimethylation of histone H3 lysine 27 is induced by chronic inflammation in mouse colonic epithelial cells, and is involved in the formation of a field for cancerization. [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 5349. doi:10.1158/1538-7445.AM2013-5349
Abstract 5358: BRCA1 is a negative modulator of the PRC2 complex.
Authors: Zeng Xianzhou , Chen Shuai , Wang Lan , Huang Haojie
Published: August 2014

The Polycomb repressive complex 2 (PRC2) is important for maintenance of stem cell pluripotency and suppression of cell differentiation by promoting histone H3 lysine 27 trimethylation (H3K27me3) and transcriptional repression of differentiation regulatory genes. Here we show that the tumor suppressor protein BRCA1 interacts with EZH2 and other components of PRC2 in mouse embryonic stem (ES) and human breast cancer cells. The BRCA1-binding region in EZH2 overlaps with the non-coding RNA (ncRNA)-binding domain, and BRCA1 expression inhibits the binding of EZH2 to the HOTAIR ncRNA. Decreased expression of BRCA1 causes EZH2 re-targeting genome wide and elevates H3K27me3 levels at PRC2 target loci in both mouse ES and human breast cancer cells. BRCA1 deficiency blocks ES cell differentiation and enhances breast cancer migration and invasion in an EZH2-dependent manner. These results reveal that BRCA1 is a key negative modulator of PRC2 and that loss of BRCA1 inhibits ES cell differentiation and enhances cancer aggressive phenotypes via augmented PRC2 function.

Citation Format: Xianzhou Zeng, Shuai Chen, Lan Wang, Haojie Huang. BRCA1 is a negative modulator of the PRC2 complex. [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 5358. doi:10.1158/1538-7445.AM2013-5358
Abstract 5373: Genome-wide methylation analysis of prostate tissues reveals global methylation patterns of prostate cancer.
Authors: Luo Jian-Hua , Ding Ying , Chen Rui , Michalopoulos George K. , Nelson Joel B. , Tseng George C. , Yu Yan P.
Published: August 2014

Altered genome methylation is a hallmark of human malignancies. To investigate the global methylation profile of prostate cancer, high-throughput analysis of concordant methylation and gene expression events was carried out for 124 human prostate specimens, including prostate cancer (T), normal adjacent to tumor (AT), and organ donor (OD). Methylated DNA in genomic DNA was immunoprecipitated with anti-methylcytidine antibodies, amplified by adaptor-specific priming PCR, and detected by Affymetrix human whole genome SNP 6.0 chips. Pair-wise analyses of methylation-enriched DNA, along with the matched genome copy number analysis of the same samples, were performed to determine the differential DNA methylation profiles of the T, AT and OD samples. A total of 34,413 methylated genome segments were identified. Among these, 11,481 CpG islands were found in the promoter and exon 1 regions of 9,295 genes. Seven-thousand-six-hundred-forty-one genes were found to be methylated frequently across the OD, AT and T samples, while 239 genes were found to be differentially methylated in only T and 785 genes were methylated in both AT and T but not OD. Genes with promoter methylation and concordant suppressed expression were identified by the pair analyses of T/OD, AT/OD, and T/AT. Pathway analysis suggested that many of the methylated genes in T and AT had cell growth and mitogenic functions. Classification analysis of the differentially methylated genes in T or OD produced a specificity of 89.4% and a sensitivity of 85.7%. The T and AT groups, on the other hand, were only slightly separated by the prediction analysis, indicating a strong field effect. A gene methylation prediction model was developed using the methylation status of 20 genes, and was shown to predict prostate cancer relapse with a sensitivity of 80.0% and a specificity of 85.0%. Collectively, these results suggest methylation patterns may be useful predictive markers of prostate cancer clinical outcomes.

Citation Format: Jian-Hua Luo, Ying Ding, Rui Chen, George K. Michalopoulos, Joel B. Nelson, George C. Tseng, Yan P. Yu. Genome-wide methylation analysis of prostate tissues reveals global methylation patterns of prostate 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 5373. doi:10.1158/1538-7445.AM2013-5373

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 5379: Recurrent inactivation of PARD3, a polarity-related gene, in squamous cell carcinomas of the lung.
Authors: Bonastre Ester , Zondervan Ilse , Facchinetti Federica , Condom Enric , Savola Suvi , Carretero Julian , Sidransky David , Roz Luca , Brambilla Elisabeth , Sanchez-Cespedes Montse
Published: August 2014

In spite of the recent advances in cancer genomics, the genetics underlying the development of lung squamous cell carcinomas (SCC) is still poorly understood. Here, we investigated the contribution of the cell polarity-related gene, PARD3, to lung SCC carcinogenesis. First, we tested for PARD3 alterations in lung cancer cell lines from various histopathological types. The analysis confirmed an intragenic deletion at the H157 cells and unveiled biallelic mutations in another cell line. Both cell lines are SCCs, which circumscribed PARD3 alterations to this lung cancer type. Next, we extended the genetic screening, which included the determination of mutations and of intragenic deletions by multiplex ligation-dependent probe amplification (MLPA) assay, to lung primary SCCs. Overall, our analysis found that eight per cent of the 125 lung SCCs tested, endured biallelic and tumor-specific alterations at PARD3. The absence of PAR3 protein was evident in those tumors carrying frameshift-type mutations or intragenic deletions, while abnormally high immunostaining of PAR3 was observed in some PARD3 wild type tumors, suggesting positive feedback mechanisms. Further, we demonstrate that, as compared to the wild type, PAR3 mutant proteins were not able to significantly reduce cell growth, restore the appropriate formation of tight junctions or regulate downstream targets, evidencing the lack of function of these mutations. In conclusion, here we report, for the first time, the presence of recurrent and deleterious mutations at PARD3 in lung primary SCCs, which attest to its relevance in the development of lung SSCs.

Citation Format: Ester Bonastre, Ilse Zondervan, Federica Facchinetti, Enric Condom, Suvi Savola, Julian Carretero, David Sidransky, Luca Roz, Elisabeth Brambilla, Montse Sanchez-Cespedes. Recurrent inactivation of PARD3, a polarity-related gene, in squamous cell carcinomas of the lung. [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 5379. doi:10.1158/1538-7445.AM2013-5379
Abstract 5389: Novel inhibitors of nicotinamide phosphoribosyl transferase (NAMPT).
Authors: Ramachandra Murali , Pandit Chetan , Subramanya Hosahalli , Chikkanna Dinesh , Lakshminarasimhan Anirudha , Khairnar Vinayak , Panigrahi Sunil , Ramanathan Anuradha , Satyanandan Aparna , Rao Narasimha , Bera Arnab , Narayanan Kishore , Gopinath Sreevalsam , Ramachandra Raghuveer
Published: August 2014

Nicotinamide phosphoribosyl transferase (NAMPT) is the enzyme that catalyzes the rate limiting step in the salvage pathway of Nicotinamide Adenine Dinucleotide (NAD) biosynthesis. NAMPT is reported to be overexpressed in a number of cancer and inflammatory indications. Because of the requirement of NAD as a co-factor or substrate for a number of key biochemical pathways including those catalyzed by PARP1, Sirtuins and ADP-ribosyl cyclase, inhibition of NAMPT has been shown to result in anti-tumor efficacy in preclinical models. Two NAMPT Inhibitors FK866/APO866 and GMX1778 are currently in clinical trials for oncology indications. In the presence of these clinical agents, cultured cell lines show development of resistance due to mutations underscoring the potential need for inhibitors from distinct chemical series.

Here, we report a structure-guided drug design based approach for identification of lead compounds from two chemical series selectively targeting NAMPT. Determination of co-crystal structures with several de novo designed hits greatly aided in the identification of lead compounds that exhibited potent inhibition of NAMPT against both wild type and resistance mutants (G217R and H191R) Lead compounds were highly active in inhibiting proliferation that correlated well with cellular NAD depletion in several cancer cell lines. Normal cells and selected cancer cell lines have an NAMPT independent salvage pathway for biosynthesis of NAD, which is dependent on nicotinic acid phosphoribosyltransferase (NAPRT) and Nicotinic acid (NA). The anti-proliferative activities were fully rescued in NAPRT- proficient cell lines with the addition of NA, confirming the mechanism of action through specific NAD depletion. Lead compounds from both series exhibited excellent drug-like properties including solubility, metabolic stability and permeability, and desired exposure in pharmacokinetic studies. Anti-tumor activities of these compounds including NA rescue in NAPRT-proficient tumor models are currently being evaluated in preclinical models.

Citation Format: Murali Ramachandra, Chetan Pandit, Hosahalli Subramanya, Dinesh Chikkanna, Anirudha Lakshminarasimhan, Vinayak Khairnar, Sunil Panigrahi, Anuradha Ramanathan, Aparna Satyanandan, Narasimha Rao, Arnab Bera, Kishore Narayanan, Sreevalsam Gopinath, Raghuveer Ramachandra. Novel inhibitors of nicotinamide phosphoribosyl transferase (NAMPT). [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 5389. doi:10.1158/1538-7445.AM2013-5389
Abstract 54: Marked reduction of sequence artifacts in massively parallel sequencing of formalin-fixed paraffin-embedded DNA by depletion of uracil containing templates.
Authors: Do Hongdo , Wong Stephen Q. , Li Jason , Dobrovic Alexander
Published: August 2014

Formalin-fixed and paraffin-embedded (FFPE) tissues are commonly used for the detection of mutational biomarkers in cancer patients. One of the challenges associated with FFPE DNA in genetic testing is its high number of sequence artefacts, thus increasing the risk of false positive results particularly for low level mutations. We have previously shown that uracil lesions in FFPE DNA due to deamination of cytosines are the major cause of C:G>T:A sequence artefacts and pretreatment of damaged FFPE DNA with uracil-DNA glycosylase (UDG) prior to PCR amplification markedly reduces the C:G>T:A sequence artefacts using methodologies suitable for the testing of limited numbers of genomic loci. As the number of cancer associated mutations increases, massively parallel sequencing (MPS) approaches are increasingly necessary. In particular, the use of targeted amplicon based strategies is being implemented in many laboratories. We set out to test whether sequence artifacts were a serious problem and whether the UDG pretreatment strategy is also applicable to a amplicon based targeted MPS approach (Truseq Cancer Panel).

Samples not treated with UDG showed multiple sequence artefacts with a significant portion of changes greater than background sequencing error, with C:G>T:A changes forming the vast majority of artefacts. Treatment of the samples with UDG prior to sequencing dramatically reduced the majority of C:G>T:A changes without affecting the detection of known SNPs and somatic mutations. Elimination of artefactual changes by UDG treatment also improved the accurate detection of low-level sequence variants. Interestingly, most C:G>T:A changes that occurred in the context of CpG dinucleotides were resistant to the UDG treatment as a likely consequence of 5-methyl cytosine being deaminated to thymine.

This approach will greatly facilitate accurate detection of mutations in FFPE samples using amplicon-based approaches which is imperative for personalised cancer medicine, especially when working with samples of low tumour purity. This work also has implications for choice of tissue and protocols for the assessment of heterogeneity in tumours.

Citation Format: Hongdo Do, Stephen Q. Wong, Jason Li, Alexander Dobrovic. Marked reduction of sequence artifacts in massively parallel sequencing of formalin-fixed paraffin-embedded DNA by depletion of uracil containing templates. [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 54. doi:10.1158/1538-7445.AM2013-54

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 5413: Role of NANOG in modulating cancer cell metabolism.
Authors: Yang Lifeng , Zhao Hongyun , Marini Juan C. , Jeter Collene , Tang Dean , Nagrath Deepak
Published: August 2014

Tumors and cancer cell lines are both heterogeneous populations and contain a sub-population of undifferentiated cells known as cancer stem cells (CSCs) that are able to renew, proliferate and differentiate into tumor cells. Recent studies have shown that transcription factors (TFs) that are implicated in the pluripotency maintenance of embryonic stem cells (ESCs) are over-expressed in many tumors. Origin and progression of cancer is the result of a complex network of communication between tumor initiating cells also known as cancer stem cells (CSCs) and epithelial cells. Despite the knowledge about involvement of NANOG in modulation of cancer progression, oncogenic signaling, and tumorigenesis, it is not known about how NANOG regulates metabolic fluxes and alter cancer cell's metabolism. Here, we show that NANOG can shift cancer cells metabolism from oxidative phosphorylation to glycolysis, to meet cancer cell's energy needs, redox balance, proliferation, and invasion. Our findings are based on the quantification of metabolites, related to glycolysis, the pentose phosphate pathway, the TCA and urea cycles, and amino acids and nucleotide metabolism, and comparison of these pathways fluxes under induced and uninduced NANOG expression. Our results demonstrate the metabolic targets of NANOG on cancer progression. These insights will present a unique opportunity for diagnosing and treating cancer through CSCs.

Citation Format: Lifeng Yang, Hongyun Zhao, Juan C. Marini, Collene Jeter, Dean Tang, Deepak Nagrath. Role of NANOG in modulating cancer cell 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 5413. doi:10.1158/1538-7445.AM2013-5413
Abstract 5414: AMPK regulates HER2/EGFR activity in HER2-positive breast cancer.
Authors: Jhaveri Teraneh , Woo Ju Hyung , Gabrielson Edward
Published: August 2014

The AMP-activated Protein Kinase (AMPK) is a master regulator of cell metabolism, controlling fatty acid synthesis, protein synthesis and carbohydrate metabolism. Physiological states with increased AMPK activity are associated with decreased cancer incidence, leading to interest in AMPK as a potential target for cancer therapy and prevention. To explore a possible role of AMPK modulation in breast cancer therapy, we investigated how activation of AMPK affects breast cancer cell signaling and survival. Initial experiments found that breast cancer cell lines with amplification and over-expression of HER2 (BT474, HCC1419, and SKBR3) are 2-fold to 5-fold more sensitive to cytotoxic effects of AICAR, a canonical pharmacological activator of AMPK, than breast cancer cell lines that lack HER2 amplification (MCF7 and MDA231). In parallel to this activation of AMPK, we observed dose- and time-dependent inhibitory effects on phosphorylation and activity of HER2 and EGFR in these AICAR-treated, HER2-amplified breast cancer cells, with activation of AMPK and suppression of HER2/ EGFR activity preceding commitment to cell death. To further explore how AMPK activity affects HER2/ EGFR, we stably transfected HER2-amplified breast cancer cells with constitutively active AMPKα and observed that AMPK activated by this genetic manipulation also leads to decreased HER2 and EGFR phosphorylation and associated downstream signaling as well as reduced cell growth. Finally, we found that morin, a flavinoid compound previously found to inhibit fatty acid synthesis, also activates AMPK and inhibits HER2 and EGFR signaling in parallel, further supporting the link across these pathways. Our results lead us to postulate that AMPK regulates HER2 and EGFR activity in HER2-amplified breast cancer cells, and activation of AMPK might provide therapeutic benefit in such cancers.

Citation Format: Teraneh Jhaveri, Ju Hyung Woo, Edward Gabrielson. AMPK regulates HER2/EGFR activity in HER2-positive 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 5414. doi:10.1158/1538-7445.AM2013-5414
Abstract 5427: Increased glycolysis by 14-3-3ζ-mediated LDHA up-regulation contributes to transformation of early mammary epithelial cells.
Authors: Chang Chia-Chi , Xu Jia , Wang Hai , Zhang Chenyu , Zhang Jian , Rehman Sumaiyah K. , Li Ping , Yu Dihua
Published: August 2014

The Warburg effect is a distinct cancer hallmark that shifts the metabolic pathway in cancer cells through elevated aerobic glycolysis for energy supply and biomass. However, the mechanism of early stage metabolic dysregulation still remains unclear. 14-3-3ζ was found to be over-expressed in the early stages of breast disease and cancer (Atypical ductal hyperplasia and Ductal carcinoma in situ) and in >40% of breast cancer patients, correlating with poor patient prognosis. We found that 14-3-3ζ over-expression in non-transformed human mammary epithelial cells (HMECs), MCF10A and MCF12A, positively correlated with changes in the cellular glycolytic activity (increased glucose uptake, increased lactate production and reduced oxygen consumption). Additionally, 14-3-3ζ over-expression in HMECs induced soft agar colony formation and disorganized acini in three dimensional (3D) culture. Interestingly, we identified that lactate dehydrogenase A (LDHA) was up-regulated in these 14-3-3ζ overexpressing HMECs. LDHA knockdown in 14-3-3ζ overexpressing cells reduced aerobic glycolysis, decreased soft agar colonies and partially rescued the abnormal acinar structures, indicating LDHA upregulation contributed to 14-3-3ζ-mediated aerobic glycolysis and early transformation. Taken together, our data provide new insight into metabolic deregulation during the early stages of breast cancer transformation.

Citation Format: Chia-Chi Chang, Jia Xu, Hai Wang, Chenyu Zhang, Jian Zhang, Sumaiyah K. Rehman, Ping Li, Dihua Yu. Increased glycolysis by 14-3-3ζ-mediated LDHA up-regulation contributes to transformation of early mammary epithelial 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 5427. doi:10.1158/1538-7445.AM2013-5427
Abstract 543: A loss of function Cbl-c mutation in a murine breast cancer model.
Authors: Kales Stephen C. , Ryan Philip E. , Nau Marion , Green Jeffrey E. , Lipkowitz Stanley
Published: August 2014

Receptor Tyrosine Kinase (RTK) signaling is essential for normal biological processes and inappropriate RTK activity can result in malignant transformation. Cbl proteins are a family of RING finger ubiquitin ligases that negatively regulate a variety of RTKs, including EGFR, MET, RET and components of the TCR pathway and disruption of this negative regulation can lead to tumor initiation and progression. Recent studies have identified a growing number of Cbl mutations associated with human myeloid neoplasias and epithelial tumors. Cbl-c is the most recently identified human Cbl protein and is expressed exclusively in epithelial cells. Recently, a novel Cbl-c cDNA has been isolated from a ductal carcinoma which developed in a C3(1)SV40Tag mouse, a murine model of breast cancer driven by Large T antigen. This mutant cDNA encodes a Cbl-c protein which lacks the catalytic domain of Cbl-c, thereby resembling known Cbl family mutations associated with myeoloid neoplasias. Genomic analysis of both parental and transgenic lines shows no evidence of germline mutation indicating that this mutation is likely somatic and may contribute to malignant transformation in this animal model. In overexpression studies, this mutant Cbl-c protein fails to mediate ubiquitination and degradation of activated EGFR and acts as a dominant negative protein, preventing ubiquitination of the EGFR by wild type Cbl proteins. The mutant Cbl-c protein also enhances transformation of NIH 3T3 cells by SV40 Large T antigen and through subsequent analysis of transforming capacity and signal pathway regulation, we are assessing interacting partners of Cbl-c and the potential role of this mutation in tumorigenesis. This mutant Cbl-c protein is the first functional evidence for the involvement of Cbl-c in transformation.

Citation Format: Stephen C. Kales, Philip E. Ryan, Marion Nau, Jeffrey E. Green, Stanley Lipkowitz. A loss of function Cbl-c mutation in a murine breast cancer model. [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 543. doi:10.1158/1538-7445.AM2013-543