bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 755: FK506 binding protein 51 (FKBP51) binds to p300 and acts as transcriptional co-regulator of ABCG2 gene expression in melanoma.
Authors: Romano Simona , Nappo Giovanna , Cesaro Elena , Candela Antonio , Romano Maria Fiammetta
Published: August 2014

Several lines of evidence support a role for FKBPs in chromatin modifications and cancer. Particularly for some of them, DNA association and regulation of gene expression has been demonstrated, mediated by histone- or transcription factor- chaperone activity. FKBP51 is an immunophilin that functions also as co chaperone of several proteins and receptors. We previously found this molecule has an essential role in melanoma resistance to the therapy and aggressiveness. Melanoma cells expressing stem cell markers (MCSC) produced high levels of FKBP51. In addition, the number of MCSC (identified by ABCG2 expression) was increased in FKBP51 over expressing melanoma cell lines. By converse, reduced expression of cancer stemness genes was measured in FKBP51 silenced melanoma. Since ABCG2+ cells also contained high levels of the general transcriptional co-activator, namely E1A binding protein p300, we hypotesized that FKBP51 might act as a p300 co chaperone with a role in transcriptional regulation of stemness markers. By co-IP assays, we show that FKBP51 interacts with p300 and partecipates in transcriptional complexes with Smad 2 and p300. By chromatin immunoprecipitation assays (ChIP) we demonstrate that FKBP51 binds to chromatin. We used different couples of primers to amplify different regions of the ABCG2 promoter, in FKBP51-immunoprecipitated chromatin and found enrichment of DNA fragments corresponding to a first sequence localized at 3450 bp (-3450) from the transcription start site (TSS), a second at -3219, and a third at -939. Differently, no enrichment in closest regions was found, suggesting the specificity of FKBP51 binding to ABCG2 promoter. In addition, we found p300 bound to the same regions of ABCG2 promoter, and such binding was impaired by FKBP51 downmodulation, suggesting the immunophilin regulatated the binding of p300 to DNA. In conclusion, our study supports the conclusion that FKBP51 partecipates in the transcriptional apparatus that controls expression of melanoma stemness genes, acting as co chaperone of p300.

Citation Format: Simona Romano, Giovanna Nappo, Elena Cesaro, Antonio Candela, Maria Fiammetta Romano. FK506 binding protein 51 (FKBP51) binds to p300 and acts as transcriptional co-regulator of ABCG2 gene expression in melanoma. [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 755. doi:10.1158/1538-7445.AM2013-755
Abstract 767: RalGTPase regulation of p53 and its contribution to malignant transformation.
Authors: Tecleab Awet , Zhang Xiaolei , Sebti Said M.
Published: August 2014

The regulation of the tumor suppressor p53 by Ral GTPases and its contribution to malignant transformation are not known. Here we demonstrate that the expression of K-Ras, Ral A and Ral B, but not Akt1/2 and c-Raf, is required for maintaining low levels of p53 in human cancer cells that harbor mutant K-Ras and wild type p53. Depletion of Ral A and Ral B increases p53 protein levels by increasing its stability and this results in p53-dependent up regulation of p21waf and mdm2. Furthermore, depletion of Ral A and / or Ral B leads to a p53-dependent decrease in the proportion of cells in the S-phase of the cell cycle, inhibition of anchorage-independent growth and suppression of invasion. Thus, expression of Ral proteins is critical to maintaining low levels of p53, and this appears to be critical to the ability of Ral GTPase to induce malignant transformation.

Citation Format: Awet Tecleab, Xiaolei Zhang, Said M. Sebti. RalGTPase regulation of p53 and its contribution to malignant transformation. [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 767. doi:10.1158/1538-7445.AM2013-767
Abstract 774: RECK silencing enhances hyperplastic phenotypes in epithelial cells through c-Myc and HIF-2α.
Authors: Lee You Mie , Lee Keun Byeol , Lee Sun Hee , Phuong Nguyen Minh
Published: August 2014

Downregulation of the tumor suppressor, RECK is induced by hypoxia (Lee et al., 2010). However, the role of RECK silencing in tumorigenic hyperplastic phenotype is largely unknown. Exposure of various normal epithelial cells to hypoxia resulted in significantly increased cell proliferation. Hypoxia increased diploid S phase cells, cell cycle kinase proteins such as cyclin D1 and c-Myc involved in G1/S transition. Hypoxia promoted phosphorylation of Rb protein (p-pRb), but decreased p21cip1, p27kip1 and p16ink4A. The knockdown of RECK by small interfering (si) RNA (RECKsi) promoted cell proliferation under normoxic conditions. Furthermore, siRECK produced the same expression patterns of cell cycle kinase proteins, c-Myc, and p-pRb observed under hypoxic conditions. MMP-2, MMP-9, and MT1-MMP were activated by hypoxia, and an MMP inhibitor significantly suppressed hypoxia-induced proliferation. In addition, the siRECK-transfected cells formed larger and more numerous colonies than the control cells in vitro tumorigenic assays. Nude mice injected with stably shRECK-transfected cells developed larger tumors more rapidly than those injected with control shRNA-transfected cells. Expression of HIF-2α as well as NFκB and STAT1 was significantly increased both under hypoxic conditions and in RECK-knockdown cells. Knockdown of HIF-2α diminished hypoxia-induced proliferation of epithelial cells. Taken together, these results suggest that hypoxia-induced RECK silencing increases epithelial transformation and tumorigenicity via HIF-2α activation, in which increased metalloproteinase activity may play a role. In conclusion, the suppression of RECK may be a crucial event for achieving a hyperplastic characteristics to favor early tumorigenesis by hypoxic stress.

Citation Format: You Mie Lee, Keun Byeol Lee, Sun Hee Lee, Nguyen Minh Phuong. RECK silencing enhances hyperplastic phenotypes in epithelial cells through c-Myc and HIF-2α. [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 774. doi:10.1158/1538-7445.AM2013-774
Abstract 776: Functional analysis of BRCA1 associated protein-1 (ubiquitin carboxy-terminal hydrolase) in melanoma.
Authors: Kumar Raj , Njauw Ching-Ni , Ji Zhenyu , Miao Benchun , Taylor Michael , Rajadurai Anpuchchelvi , Udayakumar Durga , Tsao Hensin
Published: August 2014

In the US alone, 44,250 men and 32,000 women are expected to be diagnosed with melanoma in 2012. Metastatic melanoma is the leading cause of skin cancer-related deaths worldwide. In the metastatic cutaneous melanoma (CM), anti-BRAF and anti-MEK agents have recently been shown to attenuate progression of the disease, though durable responses are still rare (NEJM articles). For metastatic ocular melanoma (OM), there is currently no established treatment strategy. Thus, one major unmet need in the field is the identification of novel “driver” physiologies which underpin the progression of disease and which can be leveraged for therapeutic gain. One new component in the ubiquitination pathway that has been recently implicated in the biology of both CM and OM is BRCA1 associated protein-1 (BAP1). Preliminary data from our laboratory showed that germline inactivating mutations of BAP1 predispose individuals to both CM and metastatic OM along with other neoplasms. In this study we examined the effects of BAP1 knockdown and overexpression on melanoma cell lines and primary melanocytes. BAP1 gene is mutated in melanoma and reported to be tumor suppressor, however suppression of BAP1 led to paradoxical growth inhibition, suggesting that the effects of BAP1 may be context dependent. Currently we are performing global gene expression and allele specific functional studies in an effort to identify novel substrates and other pathways impacted by BAP1. These findings may provide mechanistic insight into the role of BAP1 in melanoma pathogenesis.

Citation Format: Raj Kumar, Ching-Ni Njauw, Zhenyu Ji, Benchun Miao, Michael Taylor, Anpuchchelvi Rajadurai, Durga Udayakumar, Hensin Tsao. Functional analysis of BRCA1 associated protein-1 (ubiquitin carboxy-terminal hydrolase) in melanoma. [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 776. doi:10.1158/1538-7445.AM2013-776
Abstract 779: ARF activation is responsible for the tumor-suppressing function of p53 in basal cell carcinomas (BCCs) in Ptch1+/- mice.
Authors: Wang Grace Y. , Wood Carla , Wang Joy , Wang Lynn , Libove Eileen , Epstein Ervin
Published: August 2014

Basal cell carcinomas (BCCs) are the most common cancer among people of European ancestry. Aberrant activation of hedgehog (HH) signaling plays a pivotal role in BCC carcinogenesis. Besides mutations in genes encoding HH pathway components, mutations in the p53 gene frequently are found in human BCCs. Consistently, our previous studies have found that the loss of p53 can dramatically accelerate BCC formation in ionizing radiation (IR)-treated Ptch1+/- mice. It remains elusive how p53 is activated in BCC development. DNA damage response (DDR) and oncogenic induced stress (OIS) are two essentially independent pathways leading to p53 activation although limited evidence suggests crosstalk between these pathways. In response to OIS, ARF is activated and leads to p53 activation, functioning as a tumor suppressor, in many cancers. However, the loss of ARF, unlike loss of p53, failed to affect the development of medulloblastoma in Ptch1+/- mice (Wetmore C., et al, 2001). In the present study, we investigated whether ARF affects BCC development in the same mice.

To address this, we bred ARFGFP/GFP mice, in which ARF function is disabled and GFP is expressed under the control of the endogenous ARF promoter, with our Ptch1+/- mice to generate Ptch1+/- ARFGFP/GFP (designated as ARF mice) for comparison with Ptch1+/- K14CreER2 p53fl/fl (PF) mice. Following our standard protocol of tamoxifen injection at age 7.5 weeks to delete p53 specifically in K14-expressing keratinocytes (for PF mice) and treating both ARF and PF mice with ionizing radiation at age 8 weeks, we compared BCC development between the above 2 lines.

We find that at age 5 months, ARF mice formed numbers of microscopic BCCs comparable to those in PF mice though the latter exhibited greater mouse-to-mouse variation. More importantly, both mouse lines developed initial visible BCCs at age 5 to 7 months, which is dramatically earlier than the age 9 to 18 months for the Ptch1+/- mice (ARF+/+ and p53+/+). In addition, ARFGFP/GFP mice responded to IR-induced DNA damage in a pattern essentially similar to ARF+/+ mice, indicating an intact DDR pathway as reported in the literature.

In conclusion, our data demonstrate that ARF is involved in BCC carcinogenesis as a tumor suppressor, probably playing the major, perhaps the exclusive, role in activating p53 in this process.

Citation Format: Grace Y. Wang, Carla Wood, Joy Wang, Lynn Wang, Eileen Libove, Ervin Epstein. ARF activation is responsible for the tumor-suppressing function of p53 in basal cell carcinomas (BCCs) in Ptch1+/- mice. [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 779. doi:10.1158/1538-7445.AM2013-779
Abstract 783: Mutational status of the p53 tumor suppressor gene is associated with expression of GSTP1 allelic variants in human GBM..
Authors: Antoun Gamil R. , Ali-Osman Francis
Published: August 2014

The tumor suppressor and transcription factor, p53, regulates the transcription of a large number of genes that control key cellular functional, such as, cell cycle arrest, DNA repair, senescence and apoptosis, and its mutational inactivation of p53 has been implicated in tumor development and progression. Mutation in p53 is a common genetic abnormality in GBM and other cancers and several studies have associated wild type p53 with high tumor drug resistance. Our laboratory previously reported that the polymorphic human GSTP1 gene is a transcriptional target of and is activated by p53 via a canonical p53 binding motif located in intron 4 of the GSTP1 gene. The ability of the p53 wild type to transcriptionally activate the GSTP1 gene, thus, defines a novel mechanism of protecting the genome and enhancement tumor drug resistance. The ability of the different GSTP1 alleles to protect against the toxicity of anticancer agents plays an important factor in this GSTP1-mediated tumor drug resistance. In a previous study, we showed that p53 mutations in GBM spanned the entire p53 gene, with the majority of the mutations in the DNA binding domain and that GSTP1 expression correlated, in most cases with wild type p53. In the present study, we examined the relationship between p53-dependent GSTP1 gene activation genetic polymorphism and expression in human GBM. We sequenced the p53 gene in 52 primary human GBMs, genotyped these tumors for their GSTP1 allelic status and determined the expression of the GSTP1 gene transcripts by quantitative PCR. The results showed the GSTP1*A allele to most highly (40%) expressed in the tumors and of these 27% harbored wild-type p53. In contrast, GSTP1*C allele was not only the least prevalent allele but also had the least association with wild-type p53. GSTP1*B, as well as, heterozygous GSTP1 alleles were intermediate in their association with p53 mutational status of the tumors. The level of expression of the individual genes also correlated with the presence of wild-type p53 in inversely with mutant p53. These results demonstrate a significant impact of p53 mutational status, not only in mediating the level of GSTP1 gene expression but also that it is related to the specific allelic form of GSTP1 that is expressed. This suggest that p53 mutational status and both GSTP1 expression and allele polymorphism may interact together to mediate glioma drug resistance.

Citation Format: Gamil R. Antoun, Francis Ali-Osman. Mutational status of the p53 tumor suppressor gene is associated with expression of GSTP1 allelic variants in human GBM. [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 783. doi:10.1158/1538-7445.AM2013-783
Abstract 786: Genomic and functional characterization identify different manifestations of p53 pathway in 17p13 deletion cases of multiple myeloma.
Authors: Teoh Phaik Ju , Yan Junli , Chng Wee Joo
Published: August 2014

Multiple myeloma (MM) is an incurable hematologic malignancy. p53, being one of the most commonly mutated tumor suppressor gene in human cancer, is found to be wild type (WT) in almost 90% of MM. Nevertheless, the frequency of p53 abnormalities is more prevalent in the advanced stage of MM, suggesting its crucial role in the disease progression. Of paramount importance, hemizygous deletion of 17p13 (17p13(del)), which harbours the TP53 gene, has been identified in more than 30% of MM patients and is significantly associated with poor prognosis. To date, there is no conclusive evidence that p53 is the critical gene. Essentially, the integrity of p53 pathway in 17p13(del) cases remains obscure. This study utilized three human myeloma cell lines (HMCL) which harbour 17p13(del) encompassing TP53, as study models. Concurrently, HMCLs with various different p53 status (WT, mutation and null) were recruited as reference model. We have identified a spectrum of unique protein expression profile amongst cells with different p53 status upon induction of the pathway. Genotoxic and non-genotoxic stress confer an intact p53 pathway in WT/WT p53 cells; whereas, p53 mutant cells and p53 null cells demonstrated a defective p53 activity, indicating that functional p53 is exclusively required in triggering the downstream response. Using the WT/WT and the mutant/null profile as reference, the three 17p13(del) cell lines exhibited distinctive phenotypes. XG6, which have basal p53 expression half of that of the WT/WT cell line, displayed a partially compromised p53 pathway. Differentially, KMS18, which has a very low level of basal p53, and JJN3 which has a complete absence of basal p53, displayed a virtual abrogation of p53 response, as evidenced by defective transcriptional activities of p53 as well as resistance to growth arrest and apoptosis. Physiological investigations revealed that the remaining single copy of p53 was likely to be negatively regulated by MDM2 overexpression and epigenetic factors. In conclusion, this study has identified different behaviors in the p53 pathway in 17p13(del) cases. p53 appears to possess haploinssufficiency properties whereby hemizygous p53 loss is sufficient to render a partially defective p53 response. p53 basal level was shown to be the critical determinant of the integrity of the pathway. This has significant clinical implications as this finding can be manipulated to identify patients with different outcome and treatment response.

Citation Format: Phaik Ju Teoh, Junli Yan, Wee Joo Chng. Genomic and functional characterization identify different manifestations of p53 pathway in 17p13 deletion cases of multiple myeloma. [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 786. doi:10.1158/1538-7445.AM2013-786
Abstract 803: Genomic sequencing of meningiomas reveals oncogenic SMO and AKT1 mutations.
Authors: Brastianos Priscilla , Horowitz Peleg M. , Santagata Sandro , Jones Robert T. , McKenna Aaron , Getz Gad , Ligon Keith , Van Hummelen Paul , Ducar Matt , Raza Alina , Sunkavalli Ashwini , MacConaill Laura , Stemmer-Rachamimov Anat , Louis David N. , Hahn William C. , Dunn Ian , Beroukhim Rameen
Published: August 2014

Meningiomas are the most common primary brain tumor in the US. While many Grade I meningiomas are cured by surgery, 20% recur and are minimally responsive to systemic therapy. Meningiomas of higher grades are particularly challenging to treat and are associated with a poor prognosis. Though the tumor suppressor gene NF2 has been linked to meningiomagenesis, the spectrum of genetic changes in meningiomas remains poorly understood, particularly in the large subset of tumors without NF2 alterations. We performed whole-exome or whole-genome sequencing on 17 meningiomas and focused sequencing using a 645 gene-set on an additional 48 tumors. The majority of meningiomas exhibited simple genomes, with fewer copy-number alterations, mutations, and rearrangements compared to other adult tumors. However, several meningiomas harbored more complex patterns of copy-number changes and rearrangements including one tumor with chromothripsis. We confirmed focal NF2 inactivation in 43% of tumors. A subset of NF2-wildtype meningiomas harbored recurrent oncogenic mutations in AKT1 and the Hedgehog pathway regulator SMO. Tumors with these mutations varied histologically from their NF2-mutated counterparts, and exhibited immunohistochemical evidence of pathway activation. These results begin to define the spectrum of genetic alterations in meningiomas. Our findings of known driver oncogenic mutations including AKT1 and SMO have immediate therapeutic potential.

Citation Format: Priscilla Brastianos, Peleg M. Horowitz, Sandro Santagata, Robert T. Jones, Aaron McKenna, Gad Getz, Keith Ligon, Paul Van Hummelen, Matt Ducar, Alina Raza, Ashwini Sunkavalli, Laura MacConaill, Anat Stemmer-Rachamimov, David N. Louis, William C. Hahn, Ian Dunn, Rameen Beroukhim. Genomic sequencing of meningiomas reveals oncogenic SMO and AKT1 mutations. [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 803. doi:10.1158/1538-7445.AM2013-803
Abstract 817: Genomic evolution landscape of patient tumor derived xenograft of gastric cancer.
Authors: Yu Kun , Wong Swee Seong , Ting Jason C. , Doman Thompson N. , Yue Yong , aggarwal Amit , Donoho Gregory P. , Krumbach Rebekka , Feibig Heiner H. , kong Seong-Ho , Kim Woo-ho , Yang Han-kwang , Reinhard Christoph
Published: August 2014

Patient-derived xenograft (PDX) models have emerged as key model systems to understand the efficacy of anti-cancer agents. PDX systems have demonstrated superiority over cell lines with regards to higher histological resemblance to primary tumor, presence of stroma and also in mimicking response to therapeutic agents. However, the genomic evolution landscape of model establishment is not a well studied topic. In order to do that, we systematically collected materials from the first three passages of xenografts derived from eight gastric cancer patients from Seoul National University, Korea from 2008-2011. We subsequently generated comprehensive genomic profiles of the first three passages, parent primary tumor, and matched normal tissues for the eight patients. The genomic profiling data included whole-exome sequencing data (Agilent exome capture, paired-end sequencing on Illumina HiSeq 2000), mRNA expression (Affymetrix U133 Plus2.0), copy number data (Affymetrix SNP6), miRNA expression (Agilent miRNA array v16.0) and DNA methylation data (Illumina HumanMethylation27). The analysis is focused on two types of events: a) Cancer-related genomic changes derived from normal vs. tumor comparison, and their subsequent assessment in early passages; b) Passage-specific genomic changes derived from tumor vs. passages comparison. A further refinement of passage-specific genomic changes is done to differentiate ‘true’ passage-specific events from the ones that appear due to the difference in tumor purity that is lower in primary tumor due to the normal contamination. Overall, we describe the genomic landscape of evolution along xenograft establishment in gastric cancer and provide a comprehensive picture of genetic and genomic similarities (and differences) of xenografts to the primary tumor. This analysis will help us to better interpret the in vivo results emanating from experiments using xenografts and to translate the findings appropriately to the clinic.

Citation Format: Kun Yu, Swee Seong Wong, Jason C. Ting, Thompson N. Doman, Yong Yue, Amit aggarwal, Gregory P. Donoho, Rebekka Krumbach, Heiner H. Feibig, Seong-Ho kong, Woo-ho Kim, Han-kwang Yang, Christoph Reinhard. Genomic evolution landscape of patient tumor derived xenograft of gastric 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 817. doi:10.1158/1538-7445.AM2013-817

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 819: Targeted sequencing to detect somatic mutations, translocations and copy-number variation in human tumors simultaneously.
Authors: Van Hummelen Paul , Ducar Matthew , Jones Robert T. , Raza Alina , Sunkavalli Ashwini , Hanna Megan , Mills Adri , Adusumilli Ravali , Kumar Prateek , Schubert Laura , Breneiser Marc , Cooley Anna C. , Garcia Elizabeth , Scholl Lynette M. , Lindeman Neal I. , Wagle Nikhil , Garraway Levi , Cibulskis Kristian , Carter Scott L. , Lawrence Michael , Getz Gad , Meyerson Matthew L. , Hahn William C. , MacConaill Laura E.
Published: August 2014

The ability to harness the power of next generation sequencing for characterizing the cancer genome would be extremely valuable to the cancer research community and ultimately improve diagnosis and individualized therapy.

We present a targeted approach using massively parallel sequencing to simultaneously detect mutations, translocations and copy-number variations in archived clinical tumor specimen.

Targeted sequencing was achieved by designing RNA baits to capture the exons of 504 genes with relevance to cancer. The bait set was augmented with specific intronic sequences to detect translocations often involved in cancer. The overall performance of the hybrid capture was improved by optimizing the tiling strategy of the baits. These improvements significantly shifted fragments with low or no-coverage closer to the overall mean, resulting in a more uniform coverage distribution.

To validate the targeted gene panel we sequenced tumor samples harboring mutations, translocations and copy-number alterations that were previously identified by clinically approved assays. All the known alterations were confirmed with additional potentially actionable mutations.

The ultimate goal of the targeted panel is to screen cancer patients quickly and economically allowing simultaneous detection of all common genomic alterations in the cancer genome.

Citation Format: Paul Van Hummelen, Matthew Ducar, Robert T. Jones, Alina Raza, Ashwini Sunkavalli, Megan Hanna, Adri Mills, Ravali Adusumilli, Prateek Kumar, Laura Schubert, Marc Breneiser, Anna C. Cooley, Elizabeth Garcia, Lynette M. Scholl, Neal I. Lindeman, Nikhil Wagle, Levi Garraway, Kristian Cibulskis, Scott L. Carter, Michael Lawrence, Gad Getz, Matthew L. Meyerson, William C. Hahn, Laura E. MacConaill. Targeted sequencing to detect somatic mutations, translocations and copy-number variation in human tumors simultaneously. [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 819. doi:10.1158/1538-7445.AM2013-819
Abstract 82: CYP3A4 promotes mammary carcinoma angiogenesis in a cell iIntrinsic fashion.
Authors: Guo Zhijun , Norris Beverly , Henriksen Jonathan , Morgan Monique , Maher Michael , Schumacher Robert , Bliss Robin , Chu Haitao , Gupta Kalpna , Schmechel Stephen , Potter David
Published: August 2014

Cytochrome P450 3A4 (CYP3A4) has been recently been implicated in breast cancer cell growth and survival, but the role of cell intrinsic CYP3A4 in breast cancer cell engraftment and angiogenesis is unknown. Previously, we have characterized CYP3A4 knock down lines in which CYP3A4 is stably suppressed [J Biol Chem. 2011 May 20;286(20):17543-59]. The CYP3A4 knock down lines exhibit reduced proliferation and clonogenicity in vitro. We therefore tested in an estradiol dependent nude mouse mammary fat pad xenograft model whether the MCF-7 3-18 line (CYP3A4 shRNA), in which CYP3A4 protein levels are suppressed by 60%, exhibits reduced growth compared to a non-target line MCF-7 NT2. Before implantation, the suppression of CYP3A4 mRNA in the shRNA compared to NT2 cells was confirmed by qPCR. After 46 days of monitored tumor growth, the MCF-7 NT2 tumors exhibited a median size of 27±3 mm3 (median±SEM) compared to 2±0.6 mm3 for the MCF-7 CYP3A4 shRNA tumors. Tumor growth was modeled by a Gompertzian method and the difference was statistically significant (P=0.0018). The mammary fat pads were removed after sacrifice at the end of the experiment, inked, oriented and subjected to z-sectioning. All mice exhibited tumors. The CYP3A4 shRNA tumors exhibited necrosis (5 of 6 evaluable mice) whereas the NT2 control tumors did not (0 of 6 evaluable mice) (P=0.0152; two tailed Fisher's exact test). Angiogenesis was measured by CD31 immunohistochemical staining using an automated microvessel image analysis algorithm (Image Analysis Toolbox, Aperio Technologies) allowing measurement of percentage of area occupied by CD31 staining cells at the cortex of tumor nodules. Percent area occupied by CD31 positive cells was 2.69±0.46 vs. 0.67±0.64 (mean±SEM) (P=0.033). Together, these results suggest that cell intrinsic CYP3A4 is important for mammary carcinoma growth and angiogenesis in a xenograft model.

Citation Format: Zhijun Guo, Beverly Norris, Jonathan Henriksen, Monique Morgan, Michael Maher, Robert Schumacher, Robin Bliss, Haitao Chu, Kalpna Gupta, Stephen Schmechel, David Potter. CYP3A4 promotes mammary carcinoma angiogenesis in a cell iIntrinsic fashion. [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 82. doi:10.1158/1538-7445.AM2013-82
Abstract 822: The molecular diversity of Luminal A breast cancer.
Authors: Giovanni Ciriello , Perou Charles M. , Sander Chris , Schultz Nikolaus
Published: August 2014

Breast cancer is a collection of diseases with distinct molecular traits, prognosis, and therapeutic options. Luminal A breast cancer is the most heterogeneous, both molecularly and clinically. Using genomic data from nearly 1000 Luminal A tumors from multiple studies, we identified at least four major subtypes defined by distinct copy-number and mutation profiles. Here we show an atypical Luminal A subtype characterized by high genomic instability, TP53 mutations, and increased Aurora kinase signaling. These genomic alterations lead to a worse clinical prognosis. Copy number heterogeneity comes in tandem with distinct mutation spectra. We find a prevalence of PIK3CA, GATA3, and AKT1 mutations in the subtypes characterized by relatively diploid genomes subtypes.

Moreover, we highlight a strong association between MAP3K1 mutations and gains and losses of chromosomes 8 and 16. Finally, an unbiased pathway analysis revealed multiple rare, but mutually exclusive, alterations linked to loss of activity of co-repressor complexes N-Cor and SMRT. These rare alterations are prevalent in Luminal A tumors and may predict response to endocrine therapy. Our work shows how comprehensive genomic characterization of tumor subtypes is fundamental to inform clinical trials and to move towards personalized therapies.

Figure 1: Luminal A subtypes as determined by copy-number clustering.

Citation Format: Ciriello Giovanni, Charles M. Perou, Chris Sander, Nikolaus Schultz. The molecular diversity of Luminal A 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 822. doi:10.1158/1538-7445.AM2013-822

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 837: Identification of non-canonical NF-κB signaling as a critical mediator of Smac mimetic-stimulated migration and invasion of glioblastoma cells.
Authors: Tchoghandjian Aurelie , Jennewein Claudia , Eckhardt Ines , Rajalingam Krishna , Fulda Simone
Published: August 2014

Since inhibitor of apoptosis (IAP) proteins can regulate additional signaling pathways beyond apoptosis, we investigated the effect of the Smac mimetic BV6, which antagonizes IAP proteins, on non-apoptotic functions in glioblastoma (GBM). Here, we identify non-canonical NF-κB signaling and a TNFα/TNFR1 autocrine/paracrine loop as critical mediators of BV6-stimulated migration and invasion of GBM cells. In addition to GBM cell lines, BV6 triggers cell elongation, migration and invasion in primary, patient-derived GBM cells at non-toxic concentrations, which do not affect cell viability or proliferation, and also increases infiltrative tumor growth in vivo underscoring the relevance of these findings. Molecular studies reveal that BV6 causes rapid degradation of cIAP proteins, accumulation of NIK, processing of p100 to p52, translocation of p52 into the nucleus, increased NF-κB DNA binding and enhanced NF-κB transcriptional activity. EMSA supershift shows that the NF-κB DNA binding subunits consist of p50, p52 and RelB further confirming activation of the non-canonical NF-κB pathway. BV6-stimulated NF-κB activation leads to elevated mRNA levels of TNFα and additional NF-κB target genes involved in migration (i.e. IL-8, MCP-1, CXCR4) and invasion (i.e. MMP9). Importantly, inhibition of NF-κB by overexpression of dominant-negative IκBα superrepressor prevents the BV6-stimulated cell elongation, migration and invasion. Similarly, specific inhibition of non-canonical NF-κB signaling by RNA interference-mediated silencing of NIK suppresses the BV6-induced cell elongation, migration and invasion as well as upregulation of NF-κB target genes. Intriguingly, pharmacological or genetic inhibition of the BV6-stimulated TNFα autocrine/paracrine loop by the TNFα-blocking antibody Enbrel or by knockdown of TNFR1 abrogates the BV6-induced cell elongation, migration and invasion. By demonstrating that the Smac mimetic BV6 at non-toxic concentrations promotes migration and invasion of GBM cells via non-canonical NF-κB signaling, our findings have

Citation Format: Aurelie Tchoghandjian, Claudia Jennewein, Ines Eckhardt, Krishna Rajalingam, Simone Fulda. Identification of non-canonical NF-κB signaling as a critical mediator of Smac mimetic-stimulated migration and invasion of glioblastoma 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 837. doi:10.1158/1538-7445.AM2013-837
Abstract 846: ROS-mediated activation of AKT induces apoptosis via pVHL in prostate cancer cells .
Authors: Chetram Mahandranauth A. , Jones Kia J. , Coke Christopher , Don-Salu-Hewage Ayesha , Bethea Danaya A. , Hinton Cimona V.
Published: August 2014

Reactive oxygen species (ROS) play a central role in oxidative stress, which leads to the onset of diseases, such as cancer. Furthermore, ROS contributes to the delicate balance between tumor cell survival and death. However, the mechanisms by which tumor cells decide to elicit survival or death signals during oxidative stress are not completely understood. We have previously reported that ROS enhanced tumorigenic functions in prostate cancer cells, such as cell migration and invasion, which depended on CXCR4 and AKT signaling. Here, we report a novel mechanism by which ROS facilitated cell death through activation of AKT. We initially observed that ROS increased expression of phosphorylated AKT (p-AKT) in 22Rv1 human prostate cancer cells. The tumor suppressor PTEN, a negative regulator of AKT signaling, was rendered catalytically inactive through oxidation by ROS, although the expression levels remained consistent. Despite these events, cells still underwent apoptosis. Further investigation into apoptosis revealed that expression of the tumor suppressor pVHL increased, with concomitant decreased expression of HIF1α. Further investigation into pVHL during apoptosis revealed that the tumor suppressor becomes phosphorylated at S111 prior to activity; S111 is within a target motif (RXXS) forphosphorylation by AKT kinase. We then determined that pVHL and p-AKT associated in vitro via co-immunoprecipitation assay. Moreover, knockdown of pVHL by siRNA rescued HIF1α protein expression and rescued cells from apoptosis, which was determined by Annexin-V and cleaved-PARP. Collectively, our study suggests that in the context of oxidative stress, activatedAKT facilitated apoptosis by inducing pVHL function. Moreover, our studies suggest a novel paradigm for AKT-that it may participate in tumor suppressor rather than oncogenesis.

Citation Format: Mahandranauth A. Chetram, Kia J. Jones, Christopher Coke, Ayesha Don-Salu-Hewage, Danaya A. Bethea, Cimona V. Hinton. ROS-mediated activation of AKT induces apoptosis via pVHL in prostate 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 846. doi:10.1158/1538-7445.AM2013-846
Abstract 851: 14-3-3z-mediated suppression of tumor cell death.
Authors: Weerasekara Vajira K. , Mortenson Jeffrey B. , Andersen Joshua L.
Published: August 2014

For many cancer patients, cytotoxic chemotherapy is the primary treatment option. While the goal of these treatments is to induce tumor cell apoptosis, treatments too often fail as tumor cells possess the dynamic ability to subvert cell death and become chemoresistant. We are interested in determining the mechanisms by which tumors develop chemoresistance, with the goal of identifying potential therapeutic targets/pathways to increase the efficacy of chemotherapy. One context in which tumor cells are though to gain.14-3-3ζ is a small acidic protein that binds to phospho-serine proteins. 14-3-3ζ binding can have wide ranging effects, from the activation of enzyme function, to the orchestration of protein-protein interactions. 14-3-3ζ is highly expressed in many cancer types, and high levels of expression have been shown to correlate with the aggressiveness of tumors and lower rates of patient survival (1). However, the mechanisms by which 14-3-3ζ may promote tumor growth/tumor cell survival have yet to be determined. In this study we show that siRNA-mediated depletion of 14-3-3ζ potently sensitizes breast tumor cells to apoptosis induced by metabolic stress (e.g., hypoxia and glucose withdrawal_stresses commonly found within solid tumors) and chemotherapy. Moreover, gel filtration data suggest that metabolic cell stress induces a shift in 14-3-3ζ from high to lower molecular weight protein complexes. Drawing from these data, we performed a proteomics experiment to characterize 14-3-3ζ interactors under these conditions. We found that 14-3-3ζ associates with numerous pro-glycolysis- and cell growth-regulating enzymes (several of which are novel 14-3-3ζ interactors), and under conditions of metabolic stress, many of these interactions are diminished or lost. We are currently examining the functional consequences of these dynamic 14-3-3z interactions with respect to tumor cell survival. Together, our data suggest that 14-3-3ζ may play an apical role in regulating the metabolic response to stress, and provide insight into the mechanism by which 14-3-3ζ promotes tumor growth and survival.

Citation Format: Vajira K. Weerasekara, Jeffrey B. Mortenson, Joshua L. Andersen. 14-3-3z-mediated suppression of tumor cell death. [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 851. doi:10.1158/1538-7445.AM2013-851
Abstract 853: Inhibition of the nuclear transport protein CRM1 induces human breast cancer cell death by regulating survivin degradation.
Authors: Cheng Yan , Holloway Michael , Nguyen Kevin , Kauffman Michael , Shacham Sharon , Altura Rachel A.
Published: August 2014

Survivin is a member of the inhibitor of apoptosis protein (IAP) family based on its N-terminal baculovirus IAP repeat (BIR) domain. The protein is largely undetectable in differentiated tissues, but is highly expressed in most human tumors. Its cytoplasmic expression in these tumors correlates with reduced tumor cell death, increased resistance to cancer therapy, and decreased patient survival. Survivin is actively exported into the cytoplasm exclusively by the chromosomal region maintenance 1 (CRM1/Exportin1) protein, one of seven known nuclear export proteins. CRM1 binds to the canonical nuclear export signal (NES) domain within the survivin protein. Inhibition of CRM1-mediated nuclear export has been suggested as a novel cancer therapeutic strategy that restores the tumor suppressor function of multiple nuclear proteins. Here, we investigated the anti-tumor mechanisms of two novel, drug-like CRM1 inhibitors, KPT-185 and KPT-276, in estrogen-receptor positive and triple negative breast cancer cell lines. Tumor and control cells were treated with varying doses of the KPT compounds over time. Cell proliferation and apoptosis were measured using standard assays. Survivin localization and expression in the presence and absence of the KPT drugs was assessed by immunofluorescence microscopy and cell fractionation. Breast cancer cell lines were engineered to express higher or lower levels of Survivin to determine the contribution of Survivin inhibition to the anti-tumor effects of the CRM1 inhibitors. Results showed that the KPT compounds have potent anti-proliferative properties and that they induce time- and dose-dependent apoptosis. KPT-185 and KPT-276 initially enhanced nuclear Survivin expression but produced a decrease in total cellular Survivin levels at later time points. Survivin protein degraded at a faster rate following KPT treatment, which was blocked by treatment with proteasome inhibitors. Knockdown of Survivin increased KPT-mediated cell apoptosis while exogenous expression of Survivin rescued cells from KPT-mediated apoptosis. In summary, our data demonstrate that inhibition of nuclear export by CRM1 inhibition suppresses breast tumor cell growth and enhances tumor cell death, in part through specific targeting of the Survivin-CRM1 complex.

Citation Format: Yan Cheng, Michael Holloway, Kevin Nguyen, Michael Kauffman, Sharon Shacham, Rachel A. Altura. Inhibition of the nuclear transport protein CRM1 induces human breast cancer cell death by regulating survivin degradation. [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 853. doi:10.1158/1538-7445.AM2013-853
Abstract 889: Growth differentiation factor 15 causes increased invasiveness and EMT in HER2-overexpressing breast cancer via activation of p38 MAPK.
Authors: Joshi Jayashree P. , Chen Zhengjia , Nahta Rita
Published: August 2014

HER2 overexpression in breast cancer is frequently associated with aggressive and metastatic disease. De novo and acquired resistance to the first line therapy trastuzumab are major clinical issues that need to be addressed. We recently reported that the cytokine growth differentiation factor 15 (GDF15) is overexpressed in HER2-overexpressing breast cancer cell lines that have acquired or primary resistance to trastuzumab. GDF15, a distant member of the TGF-beta superfamily, has been associated with increased disease aggression in many malignancies. Our previous study showed that GDF15 activates the TGF-β receptor/src/HER2 signaling pathway, resulting in trastuzumab resistance. We recently observed that overexpression of GDF15 resulted in increased invasion in ovarian and breast cancer cells. In the present study, we investigate the molecular mechanisms underlying GDF15-induced invasion of HER2-overexpressing breast cancer cells resistant to trastuzumab. We demonstrate that GDF15-mediated invasion of breast cancer cells involves increased p38 MAPK activity. HER2-overexpressing breast cancer cells stably transfected with GDF15 showed EMT, increased cellular invasion, phosphorylation of p38, and increased MMP-2 and MMP-9 transcript expression. Furthermore, pharmacologic or genetic inhibition of p38 MAPK decreased invasion of breast cancer cells and improved response to trastuzumab. Also, knockdown of GDF15 suppressed expression of the p38 target genes MMP-2 and MMP-9. Finally, immunohistochemical analysis of a breast tumor tissue array showed a significant correlation (p=0.0053) between HER2 status and p38 phosphorylation in GDF15-positive breast cancers, as well as significant associations between GDF15 and MMP-2 (p=0.0027) and phosphorylated p38 and MMP-2 (0.0066) in HER2-overexpressing breast cancers. Our findings indicate a novel role for GDF15-stimulated p38 signaling in trastuzumab resistance, invasion, and tumor growth of HER2-overexpressing breast cancers.

Citation Format: Jayashree P. Joshi, Zhengjia Chen, Rita Nahta. Growth differentiation factor 15 causes increased invasiveness and EMT in HER2-overexpressing breast cancer via activation of p38 MAPK. [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 889. doi:10.1158/1538-7445.AM2013-889
Abstract 908: Identification of novel dynamic biomarkers of FGFR inhibition by the FGFR1,2 and 3 selective inhibitor AZD4547.
Authors: Delpuech Oona , Rooney Claire , Mooney Lorraine , McEwen Robert , Fenton Sarah , Baker Dawn , Shaw Robert , Dry Jonathan , Kilgour Elaine , Smith Paul
Published: August 2014

AZD4547 is an orally bio-available, highly selective and potent small molecule inhibitor, ATP competitive inhibitor of FGF receptors 1, 2 and 3. To identify novel dynamic transcript biomarkers of FGFR signalling inhibition by AZD4547, a gene expression profiling study was performed. Cell lines defined as sensitive or resistant to AZD4547 (n=10 for each class) were treated with AZD4547 or DMSO control for 2, 6 or 24 hours and processed for microarray analysis. Amongst those genes modulated by AZD4547, the MEK signature genes DUSP6 and ETV5, together with the MAPK pathway regulator SPRED1 were repressed on AZD4547 treatment across all sensitive cell lines over time, consistent with FGFR signalling through this pathway. Further in vivo validation using three FGFR sensitive/responsive xenograft models (SNU16, KMS11, KG1a) was performed. We observed time dependent repression (16h to 24h) of DUSP6, ETV5 and SPRED1 across all three xenograft models which was consistent with plasma exposure of AZD4547 in vivo. In addition, modulation of these transcript biomarkers corresponded with inhibition of phosphorylation of FGFR, FRS2 and ERK, supporting engagement of the FGFR signalling pathway. Taken together, these data identify novel dynamic biomarkers of FGFR inhibition in vivo.

Citation Format: Oona Delpuech, Claire Rooney, Lorraine Mooney, Robert McEwen, Sarah Fenton, Dawn Baker, Robert Shaw, Jonathan Dry, Elaine Kilgour, Paul Smith. Identification of novel dynamic biomarkers of FGFR inhibition by the FGFR1,2 and 3 selective inhibitor AZD4547. [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 908. doi:10.1158/1538-7445.AM2013-908