bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2792: Polymorphisms in vitamin D-related genes and risk of breast cancer
Authors: Shi Joy , Grundy Anne , Richardson Harriet , Brooks-Wilson Angela , Spinelli John , Aronson Kristan J.
Published: August 2015

Vitamin D is hypothesized to decrease breast cancer risk by controlling cell proliferation, differentiation and apoptosis, and estrogen synthesis and signalling in breast tissue. Transport of circulating vitamin D to sites of action is facilitated by vitamin D binding protein, encoded by the GC gene, whereupon it binds to the vitamin D receptor, encoded by the VDR gene. Given that polymorphisms in the GC and VDR gene have not been consistently associated with breast cancer risk, our objective was to assess the associations of vitamin D-related genes with breast cancer risk, accounting for ethnicity and menopausal status, and to determine these associations according to tumour receptor status defined by expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER2). A case-control study of 1,037 breast cancer cases and 1,050 controls age-frequency matched in Vancouver, British Columbia and Kingston, Ontario was used. The distribution and associations of 21 single nucleotide polymorphisms (SNPs) of the GC and VDR gene with breast cancer risk and breast tumour subtypes were investigated among premenopausal and postmenopausal European and Asian women in stratified analyses. Following false discovery rate adjustment, no SNP was associated with breast cancer risk among any subgroup. However, among European women, two SNPs in the VDR gene, rs1544410 (OR = 0.38, 95% CI: 0.21 - 0.70) and rs7967152 (OR = 2.80, 95% CI: 1.62 - 4.85) were associated with risk of ER-/PR-/HER2+ breast tumour subtype, but not with other breast tumour subtypes. Breast tumour subtypes are proposed to be etiologically distinct disease states, with different risk factors and prognostic outcomes. Interactions between variations in vitamin D-related genes and breast tumour subtypes are biologically plausible given our current understanding of anticarcinogenic properties of vitamin D elicited through the regulation of cell growth and reproductive hormone synthesis. These findings provide additional insight into the etiologic differences among breast tumour subtypes.

Citation Format: Joy Shi, Anne Grundy, Harriet Richardson, Angela Brooks-Wilson, John Spinelli, Kristan J. Aronson. Polymorphisms in vitamin D-related genes and risk of breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2792. doi:10.1158/1538-7445.AM2015-2792
Abstract 28: Deregulation of NAC complex inhibits muscle differentiation and blocks apoptosis in rhabdomyosarcoma cells
Authors: Zhang Meiling , Davie Judith
Published: August 2015

Rhabdomyosarcoma (RMS) is a common malignant soft tissue sarcoma in children with limited treatment options and high failure rate during standard therapy. New therapeutic targets or strategies are therefore in high demand. The NAC complex performs many diverse biological functions, and the deregulation of its subunits has been correlated with many cancers. We sought to understand the function of NAC complex in normal muscle cells and rhabdomyosarcoma cells. With western blot, quantitative real time PCR (qRT-PCR), chromatin immunoprecipitation and immunostaining analysis, we found that the muscle specific subunit of NAC complex, skNAC, which is the alternatively spliced isoform of NACα, was downregulated in RMS cells. In normal cells, skNAC shuttled from the cytoplasm to the nucleus upon differentiation. We also showed that skNAC associated with muscle specific promoters together with BTF3 in differentiated cells, and this association is dependent on the expression of BTF3. The expression of skNAC was regulated by the splicing factor SRPK3, a serine arginine protein kinase. Overexpression of SPRK3 induced skNAC expression and muscle differentiation. We also detected the deregulation of another NAC complex subunit, NACβ (also known as BTF3). We found that BTF3 is highly overexpressed and localized in the nucleus in both RMS cell lines and human RMS tumor samples compared with normal samples. Depletion of BTF3 induced apoptosis as detected by the strong cleavage of PARP and caspase 3. BTF3 depletion also decreased the cell viability in RMS by a cologenic assay. We also confirmed that BTF3 downregulation promoted muscle gene expression and induced muscle differentiation in RMS cells. However, BTF3 played a different role in normal cells. With the deletion of BTF3 in C2C12 cells, no apoptosis signal was detected and cell differentiation was inhibited, which suggested that a precise level of BTF3 is important for normal cell function. In conclusion, we found that the NAC complex subunits, skNAC and BTF3, were deregulated in RMS cells. The restoration of skNAC by SRPK3 overexpression rescued muscle differentiation. BTF3 functioned as an anti-apoptosis factor in RMS cells and depletion of BTF3 induced apoptosis and promoted differentiation. With no apoptosis effect in normal cells, BTF3 could serve as a cancer specific therapeutic target in RMS cells.

Citation Format: Meiling Zhang, Judith Davie. Deregulation of NAC complex inhibits muscle differentiation and blocks apoptosis in rhabdomyosarcoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 28. doi:10.1158/1538-7445.AM2015-28
Abstract 2811: Silver nanoparticles protect human keratinocytes from deleterious effects of ultraviolet radiation: Implications for skin cancer chemoprevention
Authors: Arora Sumit , Tyagi Nikhil , Bhardwaj Arun , Rusu Lilia , Palanki Rohan , Singh Ajay P. , Carter James E. , Singh Seema
Published: August 2015

Skin cancer is the most common form of cancer worldwide and its incidence has been increasing at an alarming rate in the United States. A solar ultraviolet (UV) radiation, particularly UVB, is the main etiological cause of skin carcinogenesis due to its DNA damaging ability. Therefore, multidisciplinary scientific research has placed specific emphasis on finding novel ways for skin cancer chemoprevention. Here, we provide first experimental evidence for chemopreventive efficacy of silver-nanoparticles (AgNPs) against UVB-induced DNA damage and apoptosis in immortalized human epidermal keratinocytes (HaCaT). AgNPs synthesized by chemical reduction method were characterized for their physicochemical properties. The nanoparticles were spherical (> 90%) in shape, in the size range of 20-50 nm, and had net negative surface charge with an average zeta potential of - 47.7 mV. AgNPs were largely non-toxic to HaCaT cells and their pretreatment protected them from UVB-induced apoptosis. Significant reduction in cyclobutane pyrimidine dimer (CPD) formation upon UVB exposure was also observed in AgNPs-pretreated HaCaT cells. In addition, AgNPs pre-treatment leads to G1 phase arrest of cell cycle in UVB-exposed HaCaT cells thus allowing sufficient time for CPDs repair. Moreover, enhanced AgNPs internalization and localization into cytoplasmic and nuclear compartments was observed by UVB irradiated HaCaT cells. Furthermore, AgNPs pretreatment altered the expression of various genes involved in cell-cycle, apoptosis and nucleotide-excision repair in UVB-irradiated HaCaT cells. Together, these findings clearly suggest the potential utility of AgNPs as novel chemopreventive agents against UVB-induced skin carcinogenesis.

Citation Format: Sumit Arora, Nikhil Tyagi, Arun Bhardwaj, Lilia Rusu, Rohan Palanki, Ajay P. Singh, James E. Carter, Seema Singh. Silver nanoparticles protect human keratinocytes from deleterious effects of ultraviolet radiation: Implications for skin cancer chemoprevention. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2811. doi:10.1158/1538-7445.AM2015-2811
Abstract 2831: Development of selective GRP94 inhibitors for the treatment of cancer
Authors: Ochiana Stefan O. , Taldone Tony , Patel Hardik J. , Patel Pallav , Pengrong Yan , Sun Weilin , Rodina Anna , Shah Smit , Gewirth Daniel T. , Chiosis Gabriela
Published: August 2015

Glucose Regulated Protein 94 (Grp94) is an Hsp90 paralog localized in the lumen of the endoplasmic reticulum (ER) of higher eukaryotes. While the mechanisms associated with Grp94-pathogenic expression are still actively studied, the focus thus far of most cancer related studies has been primarily on the immunogenic activity of Grp94/peptide complexes and the involvement of this protein in the secretion of IGF-I and IGF-II and the regulation of Toll-like receptors and integrins. This status quo has recently changed when work from our lab has shown that in certain breast cancers the maintenance of a high density HER2 species at the plasma membrane and its associated aberrant signaling also requires Grp94, rendering these tumors highly sensitive to Grp94 inhibition. The discovery of selective and potent Grp94 inhibitors has been hindered due to the high similarity of the ATP-binding regulatory pocket of the Hsp90 paralogs. However, recent work from our lab has shown that this apparent roadblock can be overcome by using library screening and structural and computational analysis to discover purine-based molecules that show 100-fold selectivity for the Grp94 isoform. Herein, we detail for the first time the structure activity relationship of the selective purine derived Grp94 molecules. This work provides insights on how to overcome the high structural similarity of Hsp90s in the ligand binding pocket, and also reports selective and therapeutically relevant Grp94 inhibitors based on a purine scaffold. These initial inhibitors have potent activity against cancer cells providing an important platform for the development of Grp94 inhibitors with drug-like features and potential for clinical translation.

Citation Format: Stefan O. Ochiana, Tony Taldone, Hardik J. Patel, Pallav Patel, Yan Pengrong, Weilin Sun, Anna Rodina, Smit Shah, Daniel T. Gewirth, Gabriela Chiosis. Development of selective GRP94 inhibitors for the treatment of cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2831. doi:10.1158/1538-7445.AM2015-2831
Abstract 2846: Ultrasensitive detection of actionable subclones in chronic myeloid leukemia
Authors: Schmitt Michael W. , Aminov Bella , Radich Jerald P. , Loeb Lawrence A.
Published: August 2015

Targeted Abl inhibitors such as imatinib have vastly improved survival in chronic myeloid leukemia (CML). However resistance occurs in many patients, most frequently due to point mutations in the Abl kinase. At the time of diagnosis, resistance mutations may already exist in a small fraction of the malignant cells. Second-generation inhibitors have been developed which can overcome specific resistance mutations. Identification of pre-existing resistance mutations would thus have important therapeutic implications, as it could inform which therapy would result in the most durable response in a given patient.

Prior searches for pre-existing Abl mutations in CML have used methods such as allele-specific PCR or next-generation DNA sequencing; these methods are prone to background errors resulting in frequent false positive mutations. For example, next-generation sequencing has an error rate of 0.1%-1%, which results in a large number of erroneous mutations that obscure true variants. Enrichment of the Abl gene is also a major limitation. Nearly all prior reports have used PCR amplification for dozens of cycles to enrich the Abl gene prior to mutation detection; this process introduces thousands of PCR errors which make differentiation of true mutations from artifactual mutations challenging.

We have developed a protocol for DNA sequencing, Duplex Sequencing, that separately tags and sequences each of the two strands of single DNA molecules. True mutations are present at the same position in both strands and are complementary, while PCR or sequencing errors occur in only one of the strands and are not scored. Duplex Sequencing has a calculated error rate of less than one artifactual mutation per billion nucleotides sequenced. To apply Duplex Sequencing to the Abl oncogene, we established a new, extremely efficient targeted capture approach that utilizes sequential rounds of capture with biotinylated DNA probes. This protocol results in >1,000,000 fold enrichment, with up to 99.8% of reads corresponding to the Abl gene. Duplex Sequencing was carried out at high depth (>100,000 fold), resulting in an average of 2,000 duplex reads per nucleotide position. The estimated accuracy for mutation identification was 99.9999%.

We are in the process of sequencing samples from CML at the time of diagnosis and relapse to assay for drug resistance mutations below the limit of detection of conventional assays. Our approach could be extended to any targeted molecular agent for which mutation of the target confers resistance to the drug.

Citation Format: Michael W. Schmitt, Bella Aminov, Jerald P. Radich, Lawrence A. Loeb. Ultrasensitive detection of actionable subclones in chronic myeloid leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2846. doi:10.1158/1538-7445.AM2015-2846
Abstract 286: Coordinated activation of TLR8 and NLRP3 by VTX-2337 (motolimod) ignites tumoricidal NK cell activity
Authors: Dietsch Greg , Lu Haling , Yang Yi , Morishima Chihiro , Chow Laura Q. , Disis Mary L. , Hershberg Robert
Published: August 2015

VTX-2337 (motolimod) is a selective toll-like receptor 8 (TLR8) agonist in clinical development as an immunotherapy for multiple oncology indications, including squamous cell carcinomas of the head and neck (SCCHN). Activation of TLR8 has been found to enhance natural killer (NK) cell activation, increase antibody-dependent cell-mediated cytotoxicity (ADCC), and induce Th1 polarizing cytokines. Further characterization of VTX-2337 reveals the release of mature IL-1β and IL-18 from monocyte cell populations through coordinated actions on both TLR8 and NLRP3. In vitro, VTX-2337 primes monocytes to produce pro-IL-1β, pro-IL-18 and caspase 1, while activating the NLRP3 inflammasome, leading to the release of mature IL-1 family cytokines. Inflammasome activation by VTX-2337 is blocked by inhibiting caspase 1, with little impact on the production of other TLR8 induced mediators such as TNFα. The release of IL-18 stimulates NK cells, and augments other activation pathways including: FcγRIII and NKG2D receptors, resulting in IFNγ production and induction of CD107α. NLRP3 activation in vivo was confirmed by a dose-related increase in plasma levels of IL-1β and IL-18 in cynomolgus monkeys administered VTX-2337. This pathway for NK activation was subsequently assessed in SCCHN patients treated with VTX-2337 in combination with cetuximab, a clinically approved epidermal growth factor receptor (EGFR) specific monoclonal antibody (mAb). Cetuximab can activate NK cells through an interaction with FcγRIII, and facilitate ADCC of tumor cells. Preliminary data from a Phase I clinical trial found that prior to treatment, patient NK cells did not effectively activate in response to ex vivo stimulation by NKG2D or FcγRIII. However, following VTX-2337 treatment, patient NK cells become more responsive to these activating signals. These results show that TLR8 stimulation and inflammasome activation by VTX-2337, can complement FcγRIII engagement, and may augment clinical responses in SCCHN patients treated with cetuximab.

Citation Format: Greg Dietsch, Haling Lu, Yi Yang, Chihiro Morishima, Laura Q. Chow, Mary L. Disis, Robert Hershberg. Coordinated activation of TLR8 and NLRP3 by VTX-2337 (motolimod) ignites tumoricidal NK cell activity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 286. doi:10.1158/1538-7445.AM2015-286
Abstract 2864: Acetylation regulates TET2 stability and enzymatic activity
Authors: Zhang Yang , Cai Yi , Xie Wenbing , Easwaran Hariharan , Baylin Stephen B.
Published: August 2015

DNA methylation, one of the key epigenetic modifications in the mammalian genome, plays fundamental roles in development and disease. Although DNA methylation and DNA methyltransferases (DNMTs) have been studied intensively, our knowledge about DNA demethylation is just starting to emerge. The Ten-eleven translocation (TET) family of proteins are newly identified methylcytosine dioxygenases/demethylases that can oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), and promote DNA demethylation either passively through DNA replication, or actively via thymine DNA glycosylase (TDG)-mediated base excision repair. TET-mediated DNA demethylation has also been shown to play important roles in diverse biological processes and tumorigenesis.

Human TET2 is frequently mutated in myeloid malignancies, while loss of 5hmC and TETs expressions have been broadly observed in many other cancer types. Recent studies suggested that TETs may be regulated by microRNAs and calpain proteases, but, overall, the regulation of TET activities is not well outlined. Here we show, in human ovarian cancer cells, that TET2 activity may be significantly modulated by post-translational modifications. TET2 is acetylated by p300/CBP at K110/111, and this acetylation can be reversed by HDAC1/2 and SIRT1/2 deacetylases. Acetylation increases TET2 stability by protecting it from ubiquitination and proteasome-mediated degradation. Interestingly, DNMT1 physically associates with and stabilizes TET2 protein, and a TET2 acetylation deficient mutant (2KR) disrupts this association, indicating that acetylation of TET2 may stimulate DNMT1 binding which prevents TET2 from degradation. In addition, we show that acetylation increases TET2 enzymatic activity. Treatment of cells with HDAC inhibitors TSA/NAM, or knocking down HDAC1/2, significantly increases global 5hmC levels. Consistent with these latter observations, the 2KR mutant almost completely abolishes TET2 catalytic activity, and this mutant protein is less efficient in forming dimer with wild type TET2. Thus acetylation events may increase the activity of TET2 through enhanced dimerization of the protein. Taken together, our study reveals a novel acetylation-dependent regulatory mechanism controlling the function of TET2.

Citation Format: Yang Zhang, Yi Cai, Wenbing Xie, Hariharan Easwaran, Stephen B. Baylin. Acetylation regulates TET2 stability and enzymatic activity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2864. doi:10.1158/1538-7445.AM2015-2864
Abstract 2865: Characterization of histone deacetylases in embryonal rhabdomyosarcoma
Authors: Phelps Michael , Vleeshouwer-Neumann Terra , Bailey Jenna , Hickman Nicole , Chen Eleanor
Published: August 2015

Histone acetylation and deacetylation are key epigenetic modifications that influence gene expression in both normal and neoplastic cells. Histone deacetylases (HDACs) have been the target for cancer therapeutics due to their demonstrated role in modulating tumor onset and progression in a subset of cancers. We have shown that treatment with pan HDAC inhibitors leads to reduced tumor growth and increased tumor cell differentiation of cultured Embryonal Rhabdomyosarcoma (ERMS) cells. However, the role of specific HDACs in ERMS remains unknown. To determine the function specific to each HDAC in ERMS, we have employed the Clustered Regularly Interspersed Short Palindrome Repeats (CRISPR)/Cas9 nuclease-mediated genome editing technology to enable effective knockout of individual HDAC genes in ERMS. Our CRISPR/Cas9 system uses a transient and multiplex gene targeting approach that induces loss-of-function mutations with high efficiency and allows rapid phenotypic analysis. Combining this system with stable selection of mutant clones with null mutations, we have systematically targeted all class I HDAC genes in ERMS cells. Initial characterization reveals distinct functions of class I HDAC genes in ERMS tumorigenesis. HDAC1 loss-of-function in ERMS cells promotes tumor growth. By contrast, HDAC3 or HDAC8 loss-of-function reduces tumor cell viability. HDAC2 loss-of-function does not affect tumor growth but induces robust differentiation of ERMS cells. In future studies, candidate genes/pathways regulated by HDACs will be identified by an integrated approach of Chromatin Immunoprecipitation (ChIP) and gene expression profiling studies. To validate HDAC mutant phenotypes observed in vitro and assess pre-clinical efficacy of targeted HDAC therapies, CRISPR/Cas9 inducible ERMS lines are currently being developed to facilitate tumor xenograft studies in vivo. Overall, our initial gene targeting results reveal distinct roles of class I HDAC genes in modulating ERMS tumor growth and differentiation. Our results suggest that therapeutic efficacy of pan HDAC inhibitors may be compromised by simultaneously inhibiting tumor-promoting and tumor-suppressive functions of HDACs. Targeted HDAC therapies therefore, may have more potent therapeutic benefits.

Citation Format: Michael Phelps, Terra Vleeshouwer-Neumann, Jenna Bailey, Nicole Hickman, Eleanor Chen. Characterization of histone deacetylases in embryonal rhabdomyosarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2865. doi:10.1158/1538-7445.AM2015-2865
Abstract 2869: SChLAP1 mediated epigenetic modifications in prostate cancer
Authors: Singhal Udit , Sahu Anirban , Prensner John R. , Cao Qi , Chinnaiyan Arul M.
Published: August 2015

Prostate cancer is the leading cancer diagnosis in men, but only a subset of patients die from the disease. Understanding the molecular basis of aggressive prostate cancer remains elusive and few genomic biomarkers exist to guide clinical management. Long non-coding RNAs (lncRNAs) have emerged as a class of regulatory genes that play a role in several biological and disease processes, including cancer.

Recently, we identified SChLAP1, a novel, prognostic lncRNA expressed in a subset of prostate tumors. Mechanistically, SChLAP1 interacts with and inhibits genome-wide binding of the SWI/SNF nucleosome-remodeling complex. Additionally, SWI/SNF plays a key role in the epigenetic control of gene expression and has been shown to have tumor suppressive ability. Given the emerging role of SChLAP1 in the pathogenesis of aggressive prostate cancer and its interaction with the tumor suppressive SWI/SNF complex, exploring the detailed mechanism of action of SChLAP1 warranted further investigation.

To identify the regions of SChLAP1 necessary for its function, we created 250 base pair deletion constructs of SChLAP1 and measured RNA-enrichment following SWI/SNF pull-down as well as cell invasion using a Boyden chamber matrigel assay. To investigate whether SChLAP1 preferentially interacts with specific components of the SWI/SNF complex, we performed RNA immunoprecipitation assays using antibodies targeting various enzymatic subunits of SWI/SNF. SChLAP1 enrichment was measured by qPCR.

Our results indicate that there is a 250bp region of SChLAP1 that mediates its interaction with SWI/SNF and promotes an invasive phenotype. Furthermore, SChLAP1 shows binding specificity for certain SWI/SNF subunits. Future studies to elucidate the details of this interaction will provide a more comprehensive understanding of the biological and therapeutic consequences of SChLAP1 in prostate cancer progression.

Citation Format: Udit Singhal, Anirban Sahu, John R. Prensner, Qi Cao, Arul M. Chinnaiyan. SChLAP1 mediated epigenetic modifications in prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2869. doi:10.1158/1538-7445.AM2015-2869
Abstract 2881: Systemic Chromosome Instability (CIN) resulted in transcriptomic changes in metabolic and proliferation regulators in colonic mucosal tissue of Sgo1-/+ mice
Authors: Rao Chinthalapally V. , Sanghera Saira , Zhang Yuting , Biddick Laura , Reddy Arun , Lightfoot Stan , Mohammed Altaf , Dai Wei , Yamada Hiroshi Y.
Published: August 2015

(a) Colon cancer is the second most lethal cancer and is predicted to claim 50,310 lives in 2014. Chromosome Instability (CIN) is observed in 80-90% of colon cancers and is thought to contribute to colon cancer progression and recurrence. We developed animal models focusing on mitotic error-induced CIN, Shugoshin-1 (Sgo1) haploinsufficient (-/+) mice, to investigate the impact of CIN on colon cancer development. Sgo1-/+ mice showed altered profiles in colonic lesions and cancer development. In this study, we analyzed signature changes in the colonic transcriptome of Sgo1-/+ mice to investigate underlying molecular events in the altered carcinogenesis profiles.

(b) We treated control and Sgo1-/+ mice with colonic carcinogen azoxymethane (AOM), and tracked colon cancer development 12, 24, and 36 weeks after AOM treatments. We performed Next Generation Sequencing (NGS)-based transcriptome analysis at 24 weeks.

(c) There were 349 hits with 2-fold expression difference threshold, P<0.05 (217 upregulated genes, 132 downregulated genes). In normal-looking colonic mucosal tissue from Sgo1 mice, we observed notable transcriptomic changes in the lipid metabolism, Notch signaling, insulin signaling, and peroxisome proliferator-activated receptor (PPAR) pathways. The profile had partial overlap with the colon tumor transcriptome, consistent with high prevalence of CIN in colon cancer. By applying secondary selection criteria, we also identified cell surface markers that may be used to selectively target colonic CIN cells with antibody-based or vaccine approaches.

(d) Systemic Chromosome Instability resulted in transcriptomic changes in metabolic and proliferation regulators in the colon. We propose that the CIN effect may be countered through manipulation of these pathways.

Citation Format: Chinthalapally V. Rao, Saira Sanghera, Yuting Zhang, Laura Biddick, Arun Reddy, Stan Lightfoot, Altaf Mohammed, Wei Dai, Hiroshi Y. Yamada. Systemic Chromosome Instability (CIN) resulted in transcriptomic changes in metabolic and proliferation regulators in colonic mucosal tissue of Sgo1-/+ mice. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2881. doi:10.1158/1538-7445.AM2015-2881
Abstract 2886: Epigenetic regulation of MUC1 in NF-kB target gene expression of pro-inflammatory cytokines in colon cancer
Authors: Cascio Sandra , Sciurba Joshua , Finn Olivera
Published: August 2015

MUC1 is a transmembrane glycoprotein normally expressed at low levels in a fully glycosylated form on the apical surface of epithelial cells but it is found overexpressed and hypoglycosylated in human adenocarcinomas as well as sites of chronic epithelial inflammations. Aberrant glycosylation of MUC1 in cancer cells alters its normal function and affects the behavior of cancer cells. The MUC1 extracellular domain also participates in activation of the NF-kB family members such as phospho-IkBa and phospho-p65. Our data showed that the tumor form of MUC1, in association with p65, regulates the transcriptional activity of pro-inflammatory cytokines, including IL-6 and TNF-alpha, by binding to their promoter regions in both mouse and human cancer cells. Because proteomic analysis showed that MUC1 is able to interact directly with histone proteins, we hypothesized that MUC1 might influence the transcriptional activity of cytokines by inducing epigenetic modification.

Here, we explored MUC1/p65-regulated transcription of IL-6 and TNF-alpha in colon cancer cells and discovered that MUC1/p65 complex modulated the expression and interaction of histone methyltransferase Enhancer of Zeste protein-2 (EZH2) with pro-inflammatory cytokine promoters. Interestingly, the contribution of EzH2 in controlling the MUC1/p65-modulated cytokines expression was independent of its histone H3K27 methyltransferase activity but it shows novel properties in regulating chromatin remodeling that are currently under detailed investigation To investigate the in vivo significance of MUC1/p65-induced cytokines expression in the tumor microenvironment, we used human MUC1 transgenic mice that express MUC1 on the colonic epithelial cells, treated with azoxymethane to initiate and dextran sulfate sodium (AOM/DSS) to promote colorectal carcinogenesis. MUC1.Tg mice showed higher tumor incidence and decreased survival when compared with wild-type mice. Consistent with the in vitro data, NF-kB family members and EzH2 expression were higher in MUC1.Tg mice when compared to wild-type mice. In addition, the number of infiltrating macrophages strongly increased in AOM/DSS-treated colon tissues of MUC1.Tg mice. These are important findings that may explain the mechanism by which MUC1 and NF-kB regulate cancer progression.

Citation Format: Sandra Cascio, Joshua Sciurba, Olivera Finn. Epigenetic regulation of MUC1 in NF-kB target gene expression of pro-inflammatory cytokines in colon cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2886. doi:10.1158/1538-7445.AM2015-2886
Abstract 2890: Single mouse trials, a concept using patient-derived tumor xenografts for large scale in vivo screens
Authors: Gredy Christina , Schüler Julia B. , Zanella Nina , Fiebig Heinz-Herbert , Metz Thomas
Published: August 2015

Patient-derived xenografts (PDX) passaged in immunocompromised mice are a well-established system for preclinical efficacy testing of anti-cancer agents. Typically, PDXs are pre-screened either in vitro or via molecular analysis, and selected models are then tested in groups of 8 to 12 mice to confirm or evaluate the efficacy of a treatment relative to a vehicle control group. This approach results in highly reliable and reproducible efficacy data. However, it is often desirable to test large panels of tumor models in vivo in order to properly take genetic diversity into account. Such experiments can be cost-prohibitive in conventional study layouts, which results in fewer models being tested.

The Single Mouse Trial (SMT) format addresses the need for compound testing in larger, more diverse tumor populations. This format employs a single mouse per PDX model and treatment arm, thereby enabling the investigation of efficacy in substantially larger panels of PDX models. In order to better mirror inter-patient response diversity observed in the clinic, less emphasis is put on the statistical robustness of response data for individual models.

In the study presented here, six SoC drugs were tested in the SMT format in colorectal (cetuximab, oxaliplatin, irinotecan, 5-FU) and non-small-cell lung cancer (cetuximab, paclitaxel) PDXs, and results were compared to those obtained from standard format experiments with 5 - 10 mice per group. Dosing and schedules were adapted to clinical standards. Data for 19 CXF and 16 NSCLC models showed that in 77% of the cases, results obtained from the single mouse trial format were in line with results from standard efficacy tests. Furthermore, in 10% of the cases, efficacy was similar by trend between the two formats. Notable discrepancies were seen mainly in tumors exhibiting intermediate sensitivity.

Our findings indicate that for the drugs tested here, the risk of misjudging the efficacy in a given PDX model based on SMT is low. This risk could be further lowered by increasing group sizes to 3 mice. For the identification of biomarkers which requires accurate efficacy data, such an intermediate format between the SMT and the standard format might be advantageous.

Citation Format: Christina Gredy, Julia B. Schüler, Nina Zanella, Heinz-Herbert Fiebig, Thomas Metz. Single mouse trials, a concept using patient-derived tumor xenografts for large scale in vivo screens. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2890. doi:10.1158/1538-7445.AM2015-2890
Abstract 2891: Chemokine-targeted models of human orthotopic colorectal cancer in immunocompetant mice
Authors: Lipkin Steven M. , Chen Huanhuan Joyce , Sun Jian , Huang Zhiliang , Hou Harry , Arcilla Myra , Rakhilin Nikolai , Joe Daniel , Choi Jiahn , Gadamsetty Poornima , Milsom Jeff , Nandakumar Govind , Longman Randy , Zhou Kathy , Edwards Robert , Chen Kai Yuan , Bu Pengcheng , Wang Lihua , Xu Yitian , Munroe Robert , Abratte Christian , Miller Andrew , Gümüş Zeynep , Shuler Michael , Nishimura Nozomi , Edelmann Winfried , Shen Xiling
Published: August 2015

Colorectal cancer (CRC) is a leading cause of cancer death. Current human CRC orthotopic xenograft models require survival surgery and do not robustly form tumors in liver, the most common site of metastasis in patients. Here, we use chemokine-targeting to develop cell line and primary patient-derived xenograft models that recapitulate the vast majority of common human somatic CRC mutations as primary gastrointestinal (GI) tumors in mice without requiring surgery. Importantly, we utilize early-stage mouse blastocyst microinjection techniques to extend this approach and model primary human CRCs in immunoproficient mouse hosts. Next, we show that primary GI tumors can inducibly and robustly metastasize to liver. Finally, we demonstrate that human CRC liver metastases in vivo have higher levels of DKK4 and NOTCH signaling and are more chemoresistant than paired sub-cutaneous xenografts. Overall, we anticipate that this experimental system can help improve our mechanistic understanding of human primary CRC progression to liver metastasis, and augment sub-cutaneous xenografts for pre-clinical drug screening.

Citation Format: Steven M. Lipkin, Huanhuan Joyce Chen, Jian Sun, Zhiliang Huang, Harry Hou, Myra Arcilla, Nikolai Rakhilin, Daniel Joe, Jiahn Choi, Poornima Gadamsetty, Jeff Milsom, Govind Nandakumar, Randy Longman, Kathy Zhou, Robert Edwards, Kai Yuan Chen, Pengcheng Bu, Lihua Wang, Yitian Xu, Robert Munroe, Christian Abratte, Andrew Miller, Zeynep Gümüş, Michael Shuler, Nozomi Nishimura, Winfried Edelmann, Xiling Shen. Chemokine-targeted models of human orthotopic colorectal cancer in immunocompetant mice. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2891. doi:10.1158/1538-7445.AM2015-2891
Abstract 2896: A flexible mouse model of malignant pleural mesothelioma
Authors: Cooper Jonathan , Giancotti Filippo
Published: August 2015

Patients with advanced malignant pleural mesothelioma (MPM) have extremely limited treatment options. To develop targeted therapies necessary to radically alter the clinical course of MPM, it is important to identify oncogenic mutations and signaling pathways that drive its development and progression. About 75% of MPMs carry inactivating mutations at the NF2 locus, which encodes the FERM domain protein Merlin, and often also have deletions of the CDKN2A locus, encoding the CDK inhibitor p16 and the p53 activator ARF. Since traditional cancer therapies target dysregulated oncogenes, it is important to elucidate the pathogenically relevant proteins that are dysregulated downstream of MPM tumor suppressors. We have developed a flexible and rapid mouse model that can be used to dissect genetics of MPM, to evaluate the preclinical efficacy of novel therapies, and to provide a platform for MPM shRNA genetic screens. We injected into the pleural cavity of mice lentivirus encoding Cre recombinase and miR30-based shRNA cassettes to simultaneously deplete four of the most commonly lost proteins in mesothelioma - Nf2, p16INK4a/ARF, and BAP1. Cre recombinase deletes the Nf2 conditional allele and simultaneously activates a Luciferase reporter gene in the pleural mesothelium, allowing for bioluminescent imaging of infected cells that develop MPM tumors. Lentiviral miR30-based shRNA cassettes simultaneously deplete Ink4a/ARF and Bap1, helping to drive tumorigenesis. To evaluate MPM genetics, mice with conditional alleles for putative drivers of MPM pathogenesis (i.e. Dcaf1 and Ptk2) in addition to the Nf2 conditional allele can be injected with the virus to determine whether these genes are necessary and sufficient for MPM development or progression. Moreover, knockdown of putative MPM drivers can be easily carried out by adding corresponding shRNA cassettes to the lentiviral vector. Using these methods, we have developed a rapid genetically engineered mouse model that recapitulates human MPM and can be used to explore MPM genetics. This flexible MPM mouse model will allow us to unravel MPM genetics and help to identify novel therapeutic targets as well as provide an efficient preclinical test of these therapies.

Citation Format: Jonathan Cooper, Filippo Giancotti. A flexible mouse model of malignant pleural mesothelioma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2896. doi:10.1158/1538-7445.AM2015-2896
Abstract 2909: LC3 protein expression associates with UV exposure in melanoma histopathology
Authors: White Kirsten A. M. , Torres Salina , Thompson Todd A. , Hu Chien-An A. , Myers Orrin , Berwick Marianne
Published: August 2015

The 5-year survival rate for stage IV melanoma is estimated to remain unchanged at only 15%, even with new therapies. One emerging hallmark of cancer is the reprogramming of energy metabolism including the role of autophagy. In cancer, autophagy appears to be an important energy source for nutrient-depleted tumors. Ultraviolet-radiation (UVR) exposure is an established risk factor for melanoma and increases autophagic flux. We hypothesize that UV exposure is independently associated with both levels of autophagy and melanoma survival. To address this hypothesis, we are investigating the association between UVR exposure, autophagy, and development of melanoma. Basal levels of autophagy appear to vary by tumor stage, and higher levels of autophagy are associated with poor clinical prognosis. Melanoma cells contain high levels of autophagosomes, a cellular structure associated with autophagy, as measured by the proxy marker LC3. Also, the expression of the autophagy marker Beclin 1 has been associated with increased risk of early death from melanoma. These findings suggest that autophagy provides metabolic support for tumors as it localizes to hypoxic tumor areas, supports tumor cell survival, and is activated in response to stress. Autophagy can act as a cytoprotective mechanism against UV-induced apoptosis. We have shown that rates of autophagy vary by oncogene status. Autophagy was evaluated in formalin-fixed, paraffin embedded melanoma and normal tissue sections, by staining for LC3 and Beclin 1 using IHC. These markers of autophagy, along with clinical tumor characteristics, were correlated with extensive questionnaire data on UV exposure from the same patients to evaluate the impact of estimated lifetime sun exposure and sunburns on survival. A trend in the expression of Beclin1 was not observed; however, LC3 expression was increased in melanoma sections compared to normal. Our results provide enticing support that lifetime UVR exposure is associated with autophagy protein expression in melanoma.

Citation Format: Kirsten A. M. White, Salina Torres, Todd A. Thompson, Chien-An A. Hu, Orrin Myers, Marianne Berwick. LC3 protein expression associates with UV exposure in melanoma histopathology. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2909. doi:10.1158/1538-7445.AM2015-2909
Abstract 291: Targeting the stroma to hit the tumor: MMP11 as a novel target for cancer immunotherapy
Authors: Luberto Laura , Mancini Rita , Di Napoli Arianna , Peruzzi Daniela , Mori Federica , Roscilli Giuseppe , Marra Emanuele , Cappelletti Manuela , Ciliberto Gennaro , Aurisicchio Luigi
Published: August 2015

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that mediate a number of physiological and pathological processes, such as matrix degradation, tissue remodeling, inflammation and tumor metastasis. The objective of this study was to develop and characterize a vaccine targeting stromal antigens expressed by cancer associated fibroblasts (CAFs). We focused on MMP11 (or stromelysin 3, ST3), that has been detected primarily in CAFs and its expression correlates with aggressive clinical behavior and invasiveness of different types of carcinoma. We demonstrated that intramuscular injection of genetic vectors such as Adenovirus (Ad) and plasmid DNA encoding MMP11 engineered variants followed by in vivo electroporation (DNA-EP) resulted in breakage of immune tolerance and induction of both cell mediated and humoral immune response. Importantly, MMP11 vaccine was able to confer significant antitumoral protection in a chemically induced, MMP11 overexpressing colon cancer model both in prophylactic and therapeutic settings. To determine the mechanism of action of MMP11 vaccine, we have utilized IFNgamma- and μmicroMT-knock out mice, devoid of T- and B-cell immune response, respectively. Our results show that both arms of the immune response are important to confer the therapeutic effect. Moreover, MMP11 stromal vaccine showed synergic effects when combined with genetic vaccines targeting classic tumor associated antigens, such as telomerase reverse transcriptase (TERT) and carcinoembryonic antigen (CEA). Finally, to assess the immunogenicity and the safety of MMP11 vaccine in a large animal model, nonhuman primates have been vaccinated with Ad and DNA-EP. A strong immune response was measured with no detectable side effects. In addition, MMP11 detection and spontaneous immune responses in the blood of breast and prostate cancer patients further corroborate MMP11 as a valid target for immune intervention.

Taken together, these data support the use of MMP11 as a potential candidate for cancer immunotherapy in human clinical trials.

Citation Format: Laura Luberto, Rita Mancini, Arianna Di Napoli, Daniela Peruzzi, Federica Mori, Giuseppe Roscilli, Emanuele Marra, Manuela Cappelletti, Gennaro Ciliberto, Luigi Aurisicchio. Targeting the stroma to hit the tumor: MMP11 as a novel target for cancer immunotherapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 291. doi:10.1158/1538-7445.AM2015-291
Abstract 2913: Transcriptional reprogramming of pancreatic stroma induces metabolic changes in pancreatic tumor cells
Authors: Baddour Joelle , Yang Lifeng , Achreja Abhinav , Padmabandu Seth , Shah Pari , Curtis Rebecca N. , Plackemeier Thomas , Khalid Radina , Marini Juan C. , Franco-Barraza Janusz , Cukierman Edna , Hu Chaoxin , Maitra Anirban , Nagrath Deepak
Published: August 2015

Pancreatic cancer remains one of the most lethal of all solid tumors largely due to an aggressive stroma that constitutes the bulk of the tumor. The notion of a reprogrammed stroma suggests the re-establishment of a physiologically normal tumor microenvironment with quiescent stellate cells and fibroblasts. The induction of a quiescent phenotype hinders the aberrant tumor-stroma crosstalk and enables the increased intratumoral delivery of chemo drugs. A combinatorial transcriptional therapy has proven successful at normalizing the tumor microenvironment by reverting the activated state of stromal cells back to quiescence, both at the phenotypic and genetic levels. Indeed, we have observed a reduction in the activation markers of primary human stellate cells and fibroblasts, accompanied by an increase in the quiescence markers. Observations from methylation/hydroxymethylation profiling and metabolic rewiring through isotopomer flux analysis revealed potential targets for therapeutic intervention. In addition, the differential metabolic rewiring induced by a quiescent stroma in cancer cells suggests a tumor suppressive environment aimed at weakening an otherwise aggressive cancer.

Citation Format: Joelle Baddour, Lifeng Yang, Abhinav Achreja, Seth Padmabandu, Pari Shah, Rebecca N. Curtis, Thomas Plackemeier, Radina Khalid, Juan C. Marini, Janusz Franco-Barraza, Edna Cukierman, Chaoxin Hu, Anirban Maitra, Deepak Nagrath. Transcriptional reprogramming of pancreatic stroma induces metabolic changes in pancreatic tumor cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2913. doi:10.1158/1538-7445.AM2015-2913