bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 249: Breast cancer cell vesiculation is driven by calpain: implications in cancer therapy
Authors: Taylor Jack , Jaiswal Ritu , Bebawy Mary
Published: July 2016

Microparticles (MPs) are small membrane vesicles (0.1 to 1 μm in diameter) released from the plasma membranes of most cell types, (malignant and non-malignant) upon cellular activation or during apoptosis. We discovered that MPs provide a “non-genetic” mechanism for the acquisition of multidrug resistance and increased metastatic capacity in tumour cell populations, whereby they serve as vectors in the intercellular transfer of functional cancer proteins and nucleic acids. By this mechanism, MPs effectively confer the transfer, dissemination and dominance of within cancer cell populations within a matter of hours.

The aim of this study was to define the pathways governing membrane vesicle biogenesis in malignant and non-malignant cells with the aim of identifying cancer specific mediators, which could serve as novel therapeutic targets in preventing microvesicle induced deleterious traits in cancer.

We preformed a comparative analysis using the non-malignant human brain endothelial cell line (HBEC-D3) cells and drug sensitive and resistant human breast adenocarcinoma cells (MCF-7) and (MCF-7/DX) respectively. Cell surface topography and vesiculation pits were visualized at rest, and in the +/- calcium ionophore, A23187 and the Calpain inhibitor ALLM using contact mode Atomic Force Microscopy (Nanowizard, JPK Instruments, Germany) on fixed cells. We observed that at rest, cells from both malignant cell lines were high vesiculators relative to the HBEC-D3 cells. In the presence of ALLM we observed a direct inhibition of vesiculation in the malignant cells whilst vesiculation was enhanced in the non-malignant cells. Upon the release of calcium from intracellular stores using A23817, we observe an increase in vesiculation across all cell types, however to a greater extent in the HBEC-D3 cells. Interestingly, only upon the release of intracellular calcium in HBEC-D3 cells do we see an inhibition of vesiculation by ALLM. These results clearly demonstrate an alternative pathway governing vesiculation at rest for the non-malignant HBEC-D3 cells. Whereas contrary to this, vesiculation at rest in malignant cells appears to be calpain dependent. Future work aims to validate these findings and examine the presence of distinct pathways of vesiculation in malignant cells relative to non-malignant cells. These results have important implications in defining novel strategies to selectively targeting malignant cells and for the circumvention of deleterious traits acquired through intercellular exchange of extracellular vesicles.

Citation Format: Jack Taylor, Ritu Jaiswal, Mary Bebawy. Breast cancer cell vesiculation is driven by calpain: implications in cancer therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 249.
Abstract 2498: Tumorspheres cultured from circulating epithelial tumor cells (CETCs) overexpress stem cell markers in patients with solid cancers
Authors: Pachmann Katharina , Pizon Monika , Schott Dorothea , Pachmann Ulrich
Published: July 2016

Background: Solid malignancies continuously shed tumor cells which may enter the circulation, spread to other tissues and initiate metastases, but it is assumed that only a small subpopulation among CETCs is capable of metastasis formation, the subpopulation of CETCs capable of forming tumorspheres in vitro and carrying stem cells properties. The aim of this study was to characterize the pattern of expression of the tumorspheres as compared to CETCs. Methods: Blood samples were obtained from patients diagnosed with solid tumors. CETCs were enumerated with the maintrac method and subsequently cultivated in epithelial stem cell-selective medium. Immunofluorescence and qRT-PCR were performed to examine the metastatic ability of tumorspheres in vitro. Results: Analysis of surface markers in non-adherent tumorspheres forming from CETCs under stem cell- selective conditions after a period of 14 days showed typical phenotype for cancer stem cells depending on type of cancer. Furthermore, spheres had high enzymatic activity for ALDH 1. Array qRT-PCR analysis revealed that putative stem cell markers, such as Oct4, Sox2, Nanog, EpCAM, ALDH1 and CD133 are overexpressed in relation to house-keeping genes RPL13a and GAPDH in tumor spheres in contrast to the significantly lower expression level of these stem cell markers in individually isolated CETCs. High expression level of pluripotency genes in tumorspheres was associated with aggressive tumor behaviour in terms of tumor progression and type of cancer. Conclusion: Here we show that stem cell markers are overexpressed in tumorspheres as compared to CETCs. Obviously only a small population of CETCs can be grown into spheres which possess stem cell properties and most probably are responsible for recurrence and treatment failure. Precise characterization of CETCs and tumorspheres on the single cell and sphere level will contribute to a better understanding of metastasis formation and lead to development of cancer stem cell based cancer therapy.

Citation Format: Katharina Pachmann, Monika Pizon, Dorothea Schott, Ulrich Pachmann. Tumorspheres cultured from circulating epithelial tumor cells (CETCs) overexpress stem cell markers in patients with solid cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2498.
Abstract 2505: GNA13 induction of stemness and resistance to chemotherapy in breast and head and neck cancers
Authors: Leong Hui Sun , Rasheed Suhail Ahmed Kabber , Lakshmanan Manikandan , Raju Anandh Kumar , Tergaonkar Vinay , Tan Daniel shao-Weng , Casey Patrick , Iyer N.Gopalakrishna
Published: July 2016

Despite advancement in cancer therapy, the ability of cancer cells to evade therapy often leads to tumor recurrence and distant metastasis, and ultimately to the death of the patient. Increasing evidence shows that these properties of recurrence can be attributed to a small population of cells within the tumor termed cancer stem cells (CSCs). Understanding the mechanisms that support the growth of CSCs could provide new tools to target these cells and improve therapeutic outcomes. G12 proteins, comprised of Gα12 (GNA12) and Gα13 (GNA13) are mediators of G protein coupled receptor (GPCR) signaling. These proteins are highly expressed in several cancers, and blocking their signaling can significantly inhibit cancer cell invasion and metastasis. Recently, we have shown that loss of GNA13 alone can suppress invasion of breast and prostate cancer cells. Moreover, we have recently obtained evidence that enhanced expression of GNA13 is capable of inducing a stem cell phenotype in cancer cells. Specifically, enforced expression of GNA13 in cancer cells containing low levels of GNA13 protein significantly induced CSC population as shown by increased aldehyde dehydrogenase (ALDH1) activity, increased CD44+/CD24− cells, and enhancement of tumor sphere formation. In contrast, in cancer cells that express high levels of GNA13, knockdown of GNA13 suppressed ALDH1 activity, the CD44+/CD24− cell population, and the formation of spheres. Using reporter assays we have identified that GNA13 induced CSC phenotype most probably via activating Jun Kinase - AP1 signaling pathway and blocking this pathway could significantly inhibit CSC phenotype induced by GNA13. Most importantly we have identified that GNA13 expressing cells are resistant to most commonly used chemotherapeutic agents and ionizing radiations. Blocking GNA13 using shRNAs sensitized these cells to both chemo- and radiotherapy significantly. We therefore hypothesize that GNA13 plays a pivotal role in the induction of CSCs and drug resistance, and the suppression of GNA13 activity may provide therapeutic advantage to cancer therapy.

Citation Format: Hui Sun Leong, Suhail Ahmed Kabber Rasheed, Manikandan Lakshmanan, Anandh Kumar Raju, Vinay Tergaonkar, Daniel shao-Weng Tan, Patrick Casey, N.Gopalakrishna Iyer. GNA13 induction of stemness and resistance to chemotherapy in breast and head and neck cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2505.
Abstract 2514: Second generation HDAC inhibitor, Quisinostat reinstates RD3 in neuroblastoma CSCs and promotes stem cell differentiation
Authors: SOMASUNDARAM DINESH BABU , Aravindan Natarajan
Published: July 2016

Recently, we characterized the adaptive stemness and extreme plasticity of neuroblastoma (NB) cancer stem cells (CSCs). Further, new to science, we defined the loss of retinal degeneration protein 3 (RD3) in high-risk NB and identified its novel tumor evolution stabilization function. Moreover our studies identified definitive contribution of HDACs in the evolution of progressive NB. Herein, we investigated the potential of pyrimidyl-hydroxamic acid HDAC inhibitor, Quisinostat in restoring RD3 and NB-CSCs differentiation. Well characterized CD133+-CD34+ human NB CSCs exposed to increasing concentrations (2.5, 5, 10, 100, 200nM, 1, 2, 4, 8μM) of quisinostat were examined for inhibition of HDACs (HDACs 1-11, QPCR analysis), transcriptional (QPCR) and translational (immunoblotting) restoration of RD3, CSCs cell viability (automated trypan exclusion assay) differentiation (real-time live cell imaging) and formation of organized tumorospheres (tumorosphere formation assay). Quisinostat inflicted complete inhibition of NB-CSCs cell viability at 100nM concentration and beyond. QPCR analysis revealed a dose-dependent inhibition of HDACs in NB CSCs with quisinostat. We observed a significant transcriptional/translational restoration of RD3 selectively at 100nM and above of quisinostat treatment. Live-cell imaging demonstrated a dose-dependent -loss of stemness behavior and -increased differentiation of NB CSCs with quisinostat treatment. Tumorosphere formation assay demonstrated complete inhibition of organized tumorospheres at/after 100nM treatment. These results imply that Quisinostat restores RD3 and promotes NB-CSCs differentiation. More importantly, selective dose-dependent specificity of HDAC inhibition by Quisinostatnt and, restoration of RD3 and regulation of stemness physiognomies at/above 100nM concentrations identifies that HDAC 7 could serve as a critical player in this setting. Taken together, these results for the first time identify the potential of quisinostat in the regulation of NB evolution and with further studies may serve as a potential drug deliverable in the treatment and cure of high-risk NB.

Acknowledgements: Stephenson Cancer Center - Experimental Therapeutics Program, NIH COBRE 1P20GM103639-01.

Citation Format: DINESH BABU SOMASUNDARAM, Natarajan Aravindan. Second generation HDAC inhibitor, Quisinostat reinstates RD3 in neuroblastoma CSCs and promotes stem cell differentiation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2514.
Abstract 2520: IRS-1/LC3 nuclear structures and glioblastoma drug resistance
Authors: Lassak Adam , Wyczechowska Dorota , Wilk Anna , Zapata Adriana , Dean Mathew , DelValle Luis , Sarkaria Jann N. , Ochoas Augusto , Peruzzi Francesca , Reiss Krzysztof
Published: July 2016

Drug resistance and frequent tumor relapses are the major obstacles in glioblastoma therapy, and recurrent tumors are practically incurable. We previously reported that insulin receptor substrate 1 (IRS-1), which is a typical signaling molecule for insulin and insulin-like growth factor 1 receptors, can translocate to nucleus, and that nuclear IRS-1 (nIRS-1) was found in different tumor cells, including glioblastomas. To unravel its function, we employed glioblastoma cell culture, animal models, and clinical samples. Using confocal imaging, molecular cloning, subcellular fractionation, mass spectrometry, gene expression analysis, and different approaches to verify protein-protein interactions, we demonstrate for the first time that nIRS-1 can form complex nuclear structures in a restricted number of cancer cells in glioblastoma biopsies and in intracranial glioblastoma xenografts. We also demonstrated the formation of highly organized ring-like structures in several cell lines, following ectopic expression of IRS-1 cloned in frame with nuclear localization signal (NLS-IRS-1). In these nuclear structures IRS-1 localizes at the periphery, and the core of the structure harbors a key autophagy protein, LC3; however, other autophagy proteins or biological membranes were not detected. In living cells expressing NLS-IRS-1-GFP fusion protein, IRS-1/LC3 structures are highly dynamic. They rapidly exchange IRS-1 molecules with nucleoplasm and interact with other nuclear complexes including BMI1-positive Polycomb bodies, PML bodies and Cajal bodies. Importantly, clones and mixed populations of cells expressing the NLS-IRS-1 and capable of forming the IRS-1/LC3 ring-like structures undergo extensive remodeling of gene expression, which suggests a transition to stem-like phenotype and associated resistance to several different anticancer drugs, including temozolomide. This is the first demonstration of IRS-1/LC3 nuclear complexes, which are highly dynamic and may play a role in epigenetic remodeling of glioblastoma cells towards stemness. Further studies are required to determine detailed molecular composition and to explain how these new nuclear structures function.

Citation Format: Adam Lassak, Dorota Wyczechowska, Anna Wilk, Adriana Zapata, Mathew Dean, Luis DelValle, Jann N. Sarkaria, Augusto Ochoas, Francesca Peruzzi, Krzysztof Reiss. IRS-1/LC3 nuclear structures and glioblastoma drug resistance. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2520.
Abstract 2528: Mechanism and role of SOX2 repression in human germline specification
Authors: Ritu Kushwaha , Jagadish Nirmala , Bosl George , Chaganti Raju
Published: July 2016

The core pluripotency regulatory master transcription factor SOX2 is repressed in human primordial germ cells (PGCs) and in the seminoma (SEM) subset of human germ cell tumors (GCTs), which arises by transformation of PGCs. Neither the mechanism of this repression not its significance to GC and GCT development have so far been clarified. Here we show that SOX2 repression in TCam-2 SEM cells is mediated by the presence of the Polycomb repressive complex (PcG) and by the H3K27me3 chromatin mark at the SOX2 transcription start site. Moreover, repression can be abrogated by recruitment of the H3K27 demethylase UTX to the SOX2 promoter through retinoid signaling. SOX2-activated TCam-2 cells express neuronal and other lineage genes, consistent with the gene's function as a neurectodermal effector. Based on the recent discovery that SOX17 initiates human PGC specification, with its downstream target PRDM1 acting to suppress mesendodermal genes, we propose that SOX2 repression is required for PGC specification to suppress neuroectodermal genes. Overt pluripotency is initiated in latently pluripotent transformed PGCs presenting as the SOX2 upregulated embryonal carcinoma (EC) subset of GCTs; by gene expression profiling analysis we characterize the functional pathways that distinguish PGC-like unipotent SEM from blastocyst-like pluripotent EC.

Citation Format: Kushwaha Ritu, Nirmala Jagadish, George Bosl, Raju Chaganti. Mechanism and role of SOX2 repression in human germline specification. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2528.
Abstract 2534: Low glucose and hypoxia environment promote the enrichment of liver cancer stem cells
Authors: Lai Chih Chung , Shia Tsu-Hsiang , Liu Yu-Peng , Yen Chia-Hung
Published: July 2016

Cancer stem cells (CSCs), a subpopulation possess tumor initiation and self-renewal capacity, are responsible for recurrence, metastases and drug resistance. Thus, it is important to understand their biology for identification of new therapeutic approaches. Liver surgery and/or transarterial (chemo) embolization (TAE/TACE) therapy usually generate a microenvironment that fosters aggressive tumor recurrence. These areas are characterized by chronic hypoxia and possibly low glucose concentration. Here, we tested the hypothesis that whether hypoxia along with low glucose environment promote the expansion of CSC population in HCC cells. We cultured human liver cancer cell line-Huh7 cells in normoxia or hypoxia condition with high or low glucose medium. The EMT markers (N-cadherin and Vimentin) but not stemness genes (CD44 and CD133) were upregulated in hypoxia condition. However, all genes were significantly induced under low glucose/hypoxia condition. In addition, results from sphere assay showed that low glucose/hypoxia condition dramatically improved the sphere formation ability of Huh7 cells, while low glucose or hypoxia along cannot promote sphere formation. Moreover, Huh7 cells cultured in low glucose/hypoxia condition displayed a lower sensitivity to cisplatin and doxorubicin treatment compared to cells maintained in high glucose/normoxia condition. Therefore, our findings suggested that low glucose concentration and hypoxia environment remarkably promote the enrichment of liver CSCs which could be useful for developing novel therapeutic modalities.

Citation Format: Chih Chung Lai, Tsu-Hsiang Shia, Yu-Peng Liu, Chia-Hung Yen. Low glucose and hypoxia environment promote the enrichment of liver cancer stem cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2534.
Abstract 2537: Role of N-cadherin in connexin 43 mediated gap junctional formation between dormant breast cancer cells and bone marrow mesenchymal and stromal cells
Authors: Sinha Garima
Published: July 2016

Breast cancer (BC) remains a clinical problem. This is partly due to the existence of dormant BC cells (BCCs) in the bone marrow (BM) that could resurge decades after cancer remission. Cancer stem cells (CSCs) establish themselves in cycling quiescence within the BM microenvironment by forming gap junctional intercellular communication (GJIC) with mesenchymal stem cells (MSCs) and the hematopoietic supporting stroma. MiRNAs can be exchanged between the CSCs and other BM cells through the gap junction to impart cycling quiescence of the CSCs. Although the CSCs express several members of the connexin (Cx) family of proteins, GJIC between CSCs and BM cells requires Cx43. Preventing the formation of GJIC between CSCs and endogenous BM cells enhanced cycle of the BCCs with enhanced chemosensitivity. Since Cx43 is also involved in hematopoietic regulation, in order to identify targets to reverse dormancy, this study investigated the molecular mechanisms by which Cx43 is regulated, including the role of N-cadherin as a facilitator of GJIC between CSCs and BM cells. CSCs are epithelial-mesenchymal cells (EMT) and therefore express N-cadherin. In other studies Cx43 has been shown to interact with N-cadherin to facilitate the movement of Cx43 to the cell membrane. To this end, we hypothesize that the intracellular complex formed by N-cadherin and Cx43 in CSCs is regulated by the chemokine CXCL-12. MDA-MB-231, MDA-MB-468 and T47D were stimulated with different levels of CXCL12. At different times, the expression of N-cadherin was studied by real time PCR, confocal microscopy and flow cytometry. The results showed CXCL12 regulated both N-cadherin and Cx43. However, the outcome on their expression depended on the invasiveness of the tumor. Direct interaction between N-cadherin and Cx43 was demonstrated by time-line tracking for co-localization, molecular modeling and immunoprecipitation. In summary, this study showed that co-localization of N-cadherin and Cx43 is controlled by low level of CXC12. Ongoing studies are investigating how these findings affect GJIC between CSCs and BM stroma/MSCs.

Citation Format: Garima Sinha. Role of N-cadherin in connexin 43 mediated gap junctional formation between dormant breast cancer cells and bone marrow mesenchymal and stromal cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2537.
Abstract 2538: The emerging role of SOX2 in cancer cell plasticity and EGFR-TKI resistance
Authors: Chiu Yu-Fan , Kuo Ming-Han , Lee An-Chun , Chou Yu-Ting
Published: July 2016

Activating mutations in the epidermal growth factor receptor (EGFR) lead to the aberrant growth of lung adenocarcinoma while endowing cancer cells with sensitivity to EGFR-tyrosine kinase inhibitors (TKIs). Nonetheless, EGFR-TKI resistance often occurs in less than one year. The intriguing phenomenon that cancer cells have biological features similar to stem cells suggests that cancer and stem cells may share the same regulatory signaling pathway, but the involvement of stem cell signaling in EGFR-TKI resistance is not clear. Herein, we observed that SOX2, a master transcriptional factor controlling self-renewal of lung stem cells, was highly expressed in lung adenocarcinoma and inversely correlated with epithelial-to-mesenchymal transition (EMT). Clonogenic analysis showed that SOX2-silenecing attenuated cell growth in EGFR-mutated lung cancer cells. The selection of EGFR-TKI resistant HCC827 (delE746_A750) cells induced EMT while attenuating the expression of SOX2 and spheroid forming ability. Preselection of HCC827 (delE746_A750) cells with EMT features endowed cells with TKI resistance but suppressed SOX2 expression. Pharmacological inhibition of HDACs in EGFR-mutated lung cancer cells attenuated SOX2 expression but induced EMT, thus enhancing the development of EGFR-TKI resistance. Our findings support the notion that the switching expression between SOX2 and EMT plays a critical role in EGFR-TKI resistance, with the potential as biomarkers for lung cancer prediction.

Citation Format: Yu-Fan Chiu, Ming-Han Kuo, An-Chun Lee, Yu-Ting Chou. The emerging role of SOX2 in cancer cell plasticity and EGFR-TKI resistance. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2538.
Abstract 2539: Cell cycle regulation influences the phenotype of breast and prostate cancer stem cells in vitro
Authors: Feduska Joseph , Lee Carnellia , Ponnazhagan Selvarangan
Published: July 2016

The dissemination of metastases from the primary tumor accounts for over 90% of cancer-associated mortality. Despite advances on treatment modalities involving chemotherapeutic drugs, radiotherapy, and surgery, metastasis almost invariably arises by persistence of unresolved cancer cells following initial treatment. One intriguing explanation for this resiliency is the cancer stem-cell (CSC) hypothesis. According to the CSC model, phenotypic heterogeneity exists within the tumor, and a minority fraction of cells are able to reconstitute the tumor following ablation by current treatments. In like manner to their “normal” stem-cell counterparts, CSCs are capable of unlimited self-renewal, and the ability to “differentiate” to reconstitute the phenotypic heterogeneity of the initial tumor. Recent studies have sought to identify cancer stem cells in numerous types of solid tumors, including breast and prostate. Among the cell surface antigens utilized to identify the CSC subpopulations are CD24, CD44, CD133, and other factors associated with epithelial-to-mesenchymal transition (EMT).

In this study, we sought to investigate the phenotypic changes associated with cell-cycle in breast (BCa-SC) and prostate (PCa-SC) cancer. We characterized in vitro the malignant and osteolytic human breast cancer cell line MDA-MB-231 and human prostate cancer cell line PC3, with the goal of identifying an optimal checkpoint where the CSC subpopulation would be most vulnerable to subsequent targeted chemotherapy. We found that common CSC markers are expressed in a time-dependent manner due to changes in the cell cycle. Our hypothesis is that temporal inhibition of the cell cycle when the CSC phenotype is least prominent will circumvent the chemo-resistance inherent to CSC.

Citation Format: Joseph Feduska, Carnellia Lee, Selvarangan Ponnazhagan. Cell cycle regulation influences the phenotype of breast and prostate cancer stem cells in vitro. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2539.
Abstract 2559: Genome-wide haplotype association tests identified three candidate loci associated with sporadic colorectal cancer risk in Singapore Chinese
Authors: Cheah Peh Yean , Thean Lai Fun , Teo Yik Ying , Koh Woon-Puay , Yuan Jian-Min , Chew Min Hoe , Tang Choong Leong
Published: July 2016

Colorectal cancer (CRC) is one of the most frequent cancers and leading cause of cancer mortality in the developed world. Twin study has attributed about 35% of the etiology of sporadic CRC to genes. Hitherto, more than 20 single nucleotide polymorphism (SNP) loci from genome-wide association studies (GWAS) were associated with CRC risk. Most of these index SNPs however have small effect sizes and often located in gene deserts. Thus they are unlikely to be causal. Haplotype-based methods may offer a more powerful approach to disease gene mapping. We previously performed a GWAS on 2,000 ethnicity-, age-, and gender-matched Singapore Chinese CRC patients and healthy controls using the Affymetrix SNP 6 platform. Sporadic patients were defined as aged 50 or more at the time of surgery and with no dominant family history of CRC. Haplotype block detection was performed in Golden Helix SVS after this SNP6 dataset was filtered for genotype call rate (>95%), Hardy-Weinberg equilibrium in the controls and principal component analysis. Block detection was performed requiring strong linkage disequilibrium between block pairs, minor allelic frequencies (MAF) of SNPs more than 0.01, the maximum number of markers and length of the block at 30 and 160kb respectively. Genome-wide, 73,333 blocks were computed using 336, 466 markers on 22 autosomes. Haplotype frequencies were estimated using the EM algorithm. Chi-Squared and logistic regression with odds ratio tests of haplotype association were performed using these pre-computed blocks on a per-haplotype basis. Three candidate loci at chromosomes 3, 15 and 20, each with 6 to 9 SNPs and sizes ranging from 4 to 15 kb achieved genome-wide significance and encompass potential novel disease genes. These loci are currently being validated in an independent replication panel using the Fluidgm SNPtype assays and pooled analyses will be performed.

Citation Format: Peh Yean Cheah, Lai Fun Thean, Yik Ying Teo, Woon-Puay Koh, Jian-Min Yuan, Min Hoe Chew, Choong Leong Tang. Genome-wide haplotype association tests identified three candidate loci associated with sporadic colorectal cancer risk in Singapore Chinese. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2559.
Abstract 2563: Analysis of a novel gene in relation to bladder cancer GWAS signals within the 20p12.2 region
Authors: Banday Rouf , Yan Wusheng , Middlebrooks Candace D. , Kiss Eniko , Prokunina-Olsson Ludmila
Published: July 2016

Background: Genome-wide association studies (GWAS) of bladder cancer risk recently identified several signals on chromosome 20p12.2 marked by single nucleotide polymorphisms (SNPs) rs6104690, rs62185668 and rs6108803 (Figueroa et al, HMG, 2015). Interestingly, associations of SNPs rs62185668 and rs6108803 were significantly stronger for the risk of muscle-invasive (stages T2-T4) compared to non-muscle-invasive (stages Ta and T1) bladder cancer, suggesting that detailed analysis of these signals could further our understanding of aggressive bladder cancer.

Methods: We used data from The Cancer Genome Atlas (TCGA) bladder cancer set (n = 412) and an independent RNA-seq dataset for 12 bladder tissues. qRT-PCR analysis was performed with custom TaqMan assays in an additional set of 40 tumor and 40 adjacent normal bladder tissue samples. RNAscope in situ hybridization analysis with custom probes was used to evaluate mRNA expression in bladder tissue microarrays that included 7 pairs of normal-tumor bladder tissues.

Results: At a distance of more than 300 and 800 Kb, JAG1 and BTD3 are the closest genes to the GWAS signals in the 20p12.2 region. Analysis of TCGA data did not show significant association of JAG1 and BTD3 expression with genotypes of 20p12.2 GWAS markers analyzed through TCGA-genotyped proxies SNPs rs6040291 and rs6074214. Using RNA-seq data in an independent set of 12 bladder tissue samples, we discovered a novel putative gene in the 20p12.2 region. Cloning of this gene from bladder tumor tissue and annotation by several methods showed two alternative splicing forms, designated as major and minor transcripts. Expression of the major transcript in bladder tumors was significantly increased with the rs6104690-A allele (P = 0.014), which is associated with increased risk of bladder cancer. SNPs with stronger association with muscle-invasive bladder cancer (rs62185668 and rs6108803) showed weaker or non-significant associations with expression of this novel transcript (P = 0.037 and P = 0.42, respectively). This transcript might be protein-coding as it is predicted to have several open reading frames for short peptides, or it may be a long non-coding RNA (lncRNA). RNAscope analysis in bladder tumors showed nuclear expression of the major transcript. Functional analysis of this novel gene in relation to bladder cancer is underway.

Conclusions: A novel gene might be related to the GWAS signals within the 20p12.2 region and the risk of bladder cancer.

Citation Format: Rouf Banday, Wusheng Yan, Candace D. Middlebrooks, Eniko Kiss, Ludmila Prokunina-Olsson. Analysis of a novel gene in relation to bladder cancer GWAS signals within the 20p12.2 region. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2563.
Abstract 2568: Meta-analysis of genome-wide association studies identifies multiple lung cancer susceptibility loci in never-smoking Asian women
Authors: Lan Qing , Wang Zhaoming , Seow Wei Jie , Shiraishi Kouya , Shen Hongbing , Hsiung Chao A. , Matsuo Keitaro , Liu Jie , Chen Kexin , Yamji Taiki , Yang Yang , Chang I-Shou , Wu Chen , Yeager Meredith , Kohno Takashi , Zheng Wei , Shu Xiao-Ou , Yang Pan-Chyr , Wu Tangchun , Lin Dongxin , Zhou Baosen , Yu Jinming , Kubo Michiaki , Chanock Stephen J. , Rothman Nathaniel
Published: July 2016

Background: The incidence rates of lung cancer among never-smoking females in some parts of East Asia are among the highest in the world. Genome-wide association studies (GWAS) of lung cancer in Asian never-smoking women have previously identified six susceptibility loci associated with lung cancer risk including 3q28, 5p15.33, 6p21.32, 6q22.2, 10q25.2 and 17q24.3. This study aims to discover additional lung cancer susceptibility loci among never-smoking Asian females.

Methods: We imputed data from four GWAS of Asian non-smoking female lung cancer (6,877 cases and 6,277 controls) using the 1,000 Genomes Project (Phase 1 Release 3) data as the reference and genotyped additional samples (5,878 cases and 7,046 controls) for potential replication.

Results: In our meta-analysis, three new loci achieved genome-wide significance, marked by the single nucleotide polymorphisms rs7741164 at 6p21.1 (per-allele odds ratio (OR) = 1.17; P = 5.8 × 10-13), rs72658409 at 9p21.3 (per-allele OR = 0.77; P = 1.41 ×10-10), and rs11610143 at 12q13.13 (per-allele OR = 0.89; P = 4.96 ×10-9).

Conclusions: We identified new genetic susceptibility alleles for lung cancer in never-smoking women in Asia. These findings merit follow-up to understand their biological underpinnings and potential interactions with environmental exposures.

Citation Format: Qing Lan, Zhaoming Wang, Wei Jie Seow, Kouya Shiraishi, Hongbing Shen, Chao A. Hsiung, Keitaro Matsuo, Jie Liu, Kexin Chen, Taiki Yamji, Yang Yang, I-Shou Chang, Chen Wu, Meredith Yeager, Takashi Kohno, Wei Zheng, Xiao-Ou Shu, Pan-Chyr Yang, Tangchun Wu, Dongxin Lin, Baosen Zhou, Jinming Yu, Michiaki Kubo, Stephen J. Chanock, Nathaniel Rothman. Meta-analysis of genome-wide association studies identifies multiple lung cancer susceptibility loci in never-smoking Asian women. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2568.
Abstract 26: Sensitive step in cholesterol biosynthesis reveals role for sterol metabolites in regulating growth of EGFR/KRAS-dependent tumors
Authors: Gabitova Linara , Restifo Diana , Handorf Elizabeth , Cai Kathy Q. , Astsaturov Igor A.
Published: July 2016

We identified SC4MOL and NSDHL, two enzymes in the cholesterol pathway and their substrates, meiosis activating sterols (MAS), as critical regulators of receptor signaling and trafficking in normal development and in cancer. Oncogenic transformation by EGFR or RAS increases the demand for cholesterol, suggesting a possibility for metabolic interference. To test this idea in vivo, we ablated Nsdhl in adult keratinocytes expressing KRASG12D. Strikingly, Nsdhl inactivation antagonized the growth of skin tumors, while having little effect on normal skin. Loss of Nsdhl induced the expression of ATP-binding cassette (ABC) transporters ABCA1 and ABCG1, reduced the expression of low-density lipoprotein receptor (LDLR), decreased intracellular cholesterol and was dependent on the liver X receptor (LXR) α. Importantly, EGFR signaling opposed LXRα effects on cholesterol homeostasis, while an EGFR inhibitor synergized with LXRα agonists in killing cancer cells. Inhibition of SC4MOL or NSDHL, or activation of LXRα by sterol metabolites can be an effective strategy against carcinomas with activated EGFR-KRAS signaling.

Citation Format: Linara Gabitova, Diana Restifo, Elizabeth Handorf, Kathy Q. Cai, Igor A. Astsaturov. Sensitive step in cholesterol biosynthesis reveals role for sterol metabolites in regulating growth of EGFR/KRAS-dependent tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 26.
Abstract 2602: The nonclinical toxicology profile of pacritinib, a JAK2/FLT3 inhibitor with no dose-limiting clinical myelosuppression
Authors: Watson Rebecca , Al-Fayoumi Suliman
Published: July 2016

Pacritinib is an orally active kinase inhibitor being developed for the treatment of myelofibrosis (MF). Pacritinib is a particularly potent inhibitor of wild-type and mutant isoforms of JAK2 and FLT3 (IC50 values < 15 nM), and it does not suppress JAK1 at clinically relevant concentrations. Additional kinases targeted by pacritinib include IRAK1 (IC50 = 13.6 nM) and c-fms (or CSF1R; IC50 = 39.5 nM). Pacritinib is pharmacologically active in nonclinical tumor models driven by JAK2 or FLT3 overexpression, and clinical trials have demonstrated that pacritinib can ameliorate symptoms in MF patients. A key differentiating feature of pacritinib over currently available JAK2 inhibitors is its lack of dose-limiting clinical myelosuppression, allowing for treatment of patients with cytopenias. The comparatively reduced myelosuppressive activity of pacritinib is thought to be due to its lack of pharmacological activity on JAK1, and/or its inhibition of IRAK1 and c-fms, which may contribute to anti-inflammatory activity. Nonclinical pacritinib data from rodent and canine studies were evaluated in comparison to publicly available information for other JAK2 inhibitors to determine if differences in nonclinical endpoints might correlate with the observed clinical differences in myelosuppressive effects. The qualitative nonclinical findings seen in rodents were similar for all JAK2 inhibitors, and included decreases in leukocytes, red cell mass, and reticulocytes; lymphoid depletion in the spleen, thymus, and/or lymph nodes; and hypocellularity of the bone marrow. Interestingly, platelet decreases were not observed in rodent or canine nonclinical models, even with JAK2 inhibitors associated with clinical thrombocytopenia. However, in the dog, pacritinib is unique for its minimal myelosuppressive effects in the pivotal 30-day and 39-week studies, despite being performed at higher exposure levels relative to other similar JAK2 inhibitor studies. With pacritinib, non-adverse lymphoid reduction was seen in the spleen, lymph nodes, Peyer's patches, and/or thymus; but there were no clinical pathology findings indicative of marked myelosuppression, nor were there any treatment-related findings in detailed bone marrow evaluations. Overall, these comparisons suggest that nonclinical bone marrow data and clinical pathology in the dog may be informative endpoints for estimating the myelosuppressive potential of JAK2 inhibitors in the clinic.

Citation Format: Rebecca Watson, Suliman Al-Fayoumi. The nonclinical toxicology profile of pacritinib, a JAK2/FLT3 inhibitor with no dose-limiting clinical myelosuppression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2602.
Abstract 2622: A LC-MS/MS method to quantify specific dietary isothiocyanates in human urine for epidemiological studies
Authors: Dyba Marcin , Yuan Jian-Min , Adams-Haduch Jennifer , Chung Fung-Lung
Published: July 2016

Epidemiological studies have demonstrated an inverse relationship between the intake of cruciferous vegetables and risk of cancers. The protective effects may be attributed to glucosinolates and glucobrassicins in these vegetables. While glucosinolates and glucobrassicins are not chemopreventive, their metabolites, isothiocyanates (ITCs), indole-3-carbinol (I3C), and 3,3′-diindolylmethane (DIM), have been shown to inhibit tumorigenesis in rodent models. In the past studies the total ITCs intake was estimated through food frequency questionnaires and/or urinary total ITC by a HPLC-based method. Self-reported intake of cruciferous vegetables has inherent limitations in estimating the intake of ITCs because of differences in the contents of their precursors present in different kinds of cruciferous vegetables, different locations and methods of cultivation, or different methods of food preparation. Animal and cell culture studies have shown that different types of ITCs have different anticancer properties and potency, suggesting that they are not equal in protecting against the development of cancers. The lack of a reliable method to quantify specific ITCs derived from dietary intake of cruciferous vegetables hinders the investigation of the role of specific ITCs against the development of cancer in human populations. To address this deficiency, we developed a flexible, highly sensitive, fast and reliable quantitative liquid chromatography mass spectrometry method (LC-MS/MS) for detecting and quantifying common dietary ITCs plus DIM in human urine. Urinary ITCs are measured as mercapturic acid conjugates (ITCs-NAC), predominant metabolite of ITCs, using multiple reaction monitoring (MRM) technique and deuterated [2H3]-ITCs-NAC as internal standards. DIM is quantified in separate experiment using [2H2]-DIM internal standard. The method was validated by a small set of previously analyzed samples from Singapore Chinese Health Study. We found an excellent correlation between previously reported total ITCs levels and sum of ITCs detected by newly developed method. The combination of minimal sample preparation, ultra-performance liquid chromatography, and highly sensitive mass spectrometer resulted in a method that is fast (below 10 min for ITCs and 12 min for DIM) and sensitive (low femtomolar LOQ) amenable to population studies.

Citation Format: Marcin Dyba, Jian-Min Yuan, Jennifer Adams-Haduch, Fung-Lung Chung. A LC-MS/MS method to quantify specific dietary isothiocyanates in human urine for epidemiological studies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2622.
Abstract 2625A: Damnacanthal and its nanoformulation suppress cyclin D1 expression
Authors: Sukamporn Pakin , Rojanapanthu Pleumchitt , Baek Seung Joon
Published: July 2016

Damnacanthal is an anthraquinone isolated from the root of Morinda citrifolia L. (Noni) and exhibits many pharmacological properties including anti-cancer activity. It has been reported that Damnacanthal targets several signal transduction proteins related to cell growth inhibition or apoptosis. However, the molecular mechanisms by which Damnacanthal affects anti-cancer activity has not been elucidated in details. Cyclin D1 is the important regulatory protein in cell cycle progression and overexpressed in many cancer cells. In this study, we investigated the molecular mechanism of damnacanthal on cyclin D1 expression. We found that damnacanthal inhibited growth of several cancer cells (HCT-116, HT-29, MCF-7 and PC-3) in a dose- and time- dependent manner with decreasing in cyclinD1 protein expression. Damnacanthal did not change mRNA of cyclin D1, rather it suppressed cyclin D1 expression at the post-translational level. Subsequent experiments with dominant negative mutant cyclin D1 suggest that cyclin D1 at the lysine sites plays a pivotal role in damnacanthal-mediated cyclin D1 suppression. Furthermore, damnacanthal was encapsulated in self-assembled chitosan nanoparticle to improve both physicochemical and biological activities. Our results suggest that encapsulated damnacanthal exhibits better activity in cell growth inhibition, compared to non-encapsulated damnacanthal. Damnacanthal nanoformulation has potential to be a candidate for development of chemoprevention or therapeutic agent for cancers.

Grant support: This work was supported by grant from the University of Tennessee Center of Excellence in Livestock Diseases and Human Health and The Thailand Research Fund (TRF) and Faculty of Pharmacy, Mahidol University (IRG5780007).

Citation Format: Pakin Sukamporn, Pleumchitt Rojanapanthu, Seung Joon Baek. Damnacanthal and its nanoformulation suppress cyclin D1 expression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2625A.
Abstract 2627: Molecular analyses of histopathologic morphologic features in breast cancer
Authors: Heng Yu Jing Jan ,   TCGA Breast Cancer Expert Pathology Committee, Jeong Jong Cheol , Gendoo Deena M.A , Haibe-Kains Benjamin , Ciriello Giovanni , Hoadley Katherine A. , Perou Charles M. , Beck Andrew H.
Published: July 2016

Traditional histopathologic analysis of breast cancer phenotypes has played a central role in the diagnosis, prognosis and clinical management of breast cancer. This study integrated molecular data with breast cancer histopathologic annotations to elucidate the molecular basis of these common morphologic features.

We constructed a large, comprehensive histopathologic database of 850 invasive breast cancer cases from The Cancer Genome Atlas (TCGA). We integrated the consensus assessments of 11 morphologic features (nuclear pleomorphism, mitotic count, epithelial tubule formation, inflammation, DCIS, LCIS, lymphovascular invasion, necrosis, fibrotic focus, apocrine features and proportion of epithelium in invasive portion by area) with TCGA's genomic, transcriptomic and proteomic data. Using this highly annotated dataset, we identified molecular profiles associated with morphologic features, constructed Omics-based multivariate models to predict morphologic features and provided insights into their molecular etiology. The association of morphologic features’ signatures with survival in ER-positive and ER-negative breast cancer was assessed using six independent datasets. All data are publicly accessible at http://pathology.ai/tcga_breast.

Morphologic features were associated with PAM50 subtypes, PAM50 proliferation scores, genomic alterations and gene expression (p<0.05). Clustering of morphologic features and genomic alterations produced two clusters of morphologic features and their separate were driven by TP53, CDH1 and PIK3CA mutations and chr12p13.3, ch8q24.21 and chr3q26.3 amplifications. The clustering of morphologic features and gene sets/pathways also produced two clusters of morphologic features characterized by “proliferation” or “inflammation”. The transcriptomic signatures of nuclear pleomorphism and epithelial tubule formation were independently prognostic in ER-positive breast cancer. No signatures were prognostic in ER-negative.

Our detailed morphologic data enrich and complement TCGA's existing molecular data, increase our understanding of the molecular basis of breast cancer pathologic phenotypes, can facilitate the refinement of breast cancer classification, and enhance our understanding of breast cancer biology.

Citation Format: Yu Jing Jan Heng, TCGA Breast Cancer Expert Pathology Committee, Jong Cheol Jeong, Deena M.A Gendoo, Benjamin Haibe-Kains, Giovanni Ciriello, Katherine A. Hoadley, Charles M. Perou, Andrew H. Beck. Molecular analyses of histopathologic morphologic features in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2627.