bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1547: Identification of somatic mutations and copy number alterations in metastatic high-grade ovarian cancer
Authors: Lee Jung-Yun , Yoon Jung-Ki , Bang Duhee , Song Yong-Sang
Published: May 2015

Background: The extent to which metastatic tumors further evolve by accumulating additional mutations is unclear and has yet to be systematically addressed using next-generation sequencing in high-grade serous ovarian cancer.

Methods: During cytoreductive surgery prior to chemotherapy, 11 spatially separated tumor samples from primary tumor and associated metastatic sites with 2 normal samples were obtained. Whole exome sequencing and copy number analysis were performed and multiplex PCR followed by ultra-deep resequencing was used for validation. Especially, omental samples were sequenced with high depth of coverage (∼200x).Phylogenetic tree was built to explore the evolutionary relationship between samples.

Results: Only 6% of all somatic mutations were present in every sample of a given case with TP53 gene as the only known mutant gene consistently present in all samples. Two non-spatial clusters of primary samples (cluster 1 and 2), and a cluster of metastatic samples (cluster M) were identified. The patterns of mutations indicate that cluster M was originated from cluster 1, and that cluster 2 was diverged from a portion of right fimbriae in cluster 1 at the early phase of tumorigenesis. No significant metastatic sample-specific mutations or copy number changes were detected.

Conclusion: Our results showed that high level of intratumor heterogeneity was evident in a high-grade serous ovarian cancer. Moreover, we showed that transcoelomic metastasis arises with little accumulation of somatic mutations or copy number alterations.

Citation Format: Jung-Yun Lee, Jung-Ki Yoon, Duhee Bang, Yong-Sang Song. Identification of somatic mutations and copy number alterations in metastatic high-grade ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1547. doi:10.1158/1538-7445.AM2014-1547
Abstract 1550: Differential angiogenic roles of serum amyloid A 1 (SAA1) isoforms in esophageal squamous cell carcinoma (ESCC)
Authors: MAN ON YING , Lung Maria , Lung Hong Lok
Published: May 2015

Esophageal Caner (EC), a highly metastatic and fatal cancer, is ranked the eighth in mortality rate in Hong Kong cancer patients (Hong Kong Cancer Registry, Hospital Authority, 2010). Esophageal Squamous Cell Carcinoma (ESCC) is the predominant type comprising more than 90% of EC. Using a functional complementation approach, SAA1 was identified as one of the tumor suppressor gene candidates. SAA1 is located at chromosome 11p15.1 and is expressed as a secretary protein in liver, human cultured smooth muscle cells, monocyte-macrophage cell lines, and in histologically-normal human epithelial tissues. Genetic polymorphisms of SAA1 have been identified as a risk factor of diseases such as amyloidosis. Three SAA1 isoforms with two single nucleotide polymorphisms at exon three (SAA1.1, 1.3, and 1.5) were observed in the ESCC patients and healthy individuals.

The SAA proteins contain the functional YIGSR-like and RGD-like motifs, proteins with these motifs can inhibit angiogenesis, cell adhesion to ECM, growth and metastasis. To understand the anti-tumorigenic and anti-angiogenic roles of the three SAA1 isoforms in ESCC progression, both recombinant proteins and secreted proteins from the conditioned media of lentiviral-infected ESCC cell lines were used for functional assays. For the vascular endothelial cell tube formation assay, the treatments with SAA1.1 and 1.3 proteins showed suppression of tube formation, whereas no significant effects could be observed in the treatment with the SAA1.5. Suppression of cell proliferation and induction of cell death were observed when the endothelial cells were cultured with SAA1.1 and 1.3 proteins. To further elucidate the differential effects among the three SAA1 isoforms in anti-angiogenesis, the cytoskeleton arrangement of the vascular endothelial cells was studied. The SAA1.1 and SAA1.3 proteins could abolish the endothelial cell adhesion by disturbing the formation of stress fibers and focal adhesions. As a conclusion, the present data has shown the variation in anti-angiogenic potential of the three SAA1 isoforms.

We acknowledge the financial support of the Food and Health Bureau of the Hong Kong Special Administration Region, China, grant number HMRF 01120886 to HLL.

Citation Format: ON YING MAN, Maria Lung, Hong Lok Lung. Differential angiogenic roles of serum amyloid A 1 (SAA1) isoforms in esophageal squamous cell carcinoma (ESCC). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1550. doi:10.1158/1538-7445.AM2014-1550
Abstract 1551: p53 mutant regulates a network of cellular anoikis through activation of AKT and suppression of BMF
Authors: Tan Boon Shing , Tiong Kai Hung , Yap Ivan Kok Seng , Rosli Rozita , Cheong Soon-Keng , Leong Chee-Onn
Published: May 2015

p53 is the most frequently mutated tumor suppressor gene in human cancers. Unlike other tumor-suppressor genes, p53 mutations mainly occur as missense mutations within the DNA-binding domain, leading to the expression of full-length mutant p53 protein. Mutant p53 proteins not only lose their tumor suppressor function, but may also gain new oncogenic function and promote tumorigenesis through transactivation of new target genes. By using an RNA interference (RNAi) approach, we show that silencing of endogenous p53 R273H contact mutant in MDA-MB-468 cells induced massive apoptosis while no apoptosis was observed in the silencing of the p53 R175H structural mutant in SKBR3 cells. Further analysis shows that the apoptotic effects of mutant p53 knock-down involved activation of caspase-9 and mitochondria depolarisation, suggesting a role for mutant p53 in suppressing the intrinsic apoptotic pathway. Indeed, a BCL-2 family member, BCL-2 modifying factor (BMF), was reproducibly up-regulated following knock-down of mutant p53 in MDA-MB-468 cells while no significant changes was observed in other BCL2 family members. Furthermore, we show that p53 R273H contact mutant, but not p53 R175H conformation mutant, suppressed anoikis by down-regulation of BMF expression. Anoikis is a form of apoptosis induced by cell detachment and is a major mechanism to prevent metastasis. Further analyses using microarray gene profiling and connectivity map analysis show that silencing of mutant p53 in MDA-MB-468 cells generates gene expression signatures characteristic of PI3K inhibitors, suggesting that apoptotic effects of mutant p53 knock-down involved dephosphorylation of AKT, a direct substrate of PI3K. Ectopic expression of the activated form of AKT in MDA-MB-468 completely abrogated the apoptotic effects of mutant p53 knock-down. In summary, our results demonstrate that R273H mutant p53 promotes anoikis resistance in breast cancer cells that express it through suppression of BMF expression by the constitutively activated AKT pathway.

Citation Format: Boon Shing Tan, Kai Hung Tiong, Ivan Kok Seng Yap, Rozita Rosli, Soon-Keng Cheong, Chee-Onn Leong. p53 mutant regulates a network of cellular anoikis through activation of AKT and suppression of BMF. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1551. doi:10.1158/1538-7445.AM2014-1551
Abstract 1554: Identification of BRCA1 and BRCA2 somatic mutations in breast cancer tumors with loss of BRCA1 nuclear expression
Authors: Alvarez Carolina , Wiener David , Gajardo Patricia , Fernandez Wanda , Cornejo Valeria , Gamboa Jorge , Carvallo Pilar
Published: May 2015

Breast cancer is the first cause of death by cancer in women in Chile as in many countries around the world. Until today, BRCA1 and BRCA2 are the most relevant genes for breast cancer high risk. Moreover, different studies have shown that BRCA1 is under expressed in breast tumors. Our group has found that in a high percentage of hereditary tumors BRCA1 protein have a diminished or null expression, or is mislocalized to the cytoplasm (unpublished data). In this regard, it has become a goal for us to identify if mislocalization and/or loss of expression of BRCA1 and BRCA2 could be related to somatic mutations in the tumors. In this work, we sequenced BRCA1 and BRCA2 in DNA extracted from 14 fresh/frozen tumors. DNA was isolated from each tumor and amplified by PCR in 5 multiplexes (a total of 93 amplicons) considering all exons and intron/exon junctions with approximately 30bp of intronic sequence each side. Multiplexes were then pooled for each patient and processed for sequencing in the GS Junior Roche. All reads were processed with AVA software to detect nucleotide changes in BRCA1 and BRCA2 sequences and analyzed manually to discard misinterpretations. All mutations identified were confirmed by Sanger. From the same tumors we obtained a second section that was formalin fixed and paraffin embedded. This FFPE fragment was cut in 4 μm sections and analyzed by immunohistochemistry and immunofluorescence to assess BRCA1 expression and localization. We found a total of 5 mutations, 3 in BRCA1 and 2 in BRCA2, in 6 tumors. Interestingly, one BRCA1 mutation present in 7 to 20% of reads, was found in 5 tumors. Among those, two tumors presented two different mutations in BRCA2, in addition. The last tumor presented one extra mutation in BRCA1. BRCA1 expression analysis was coherent between immunohistochemistry and immunofluorescence assays. In general 9 tumors showed weak or negative BRCA1 nuclear expression and 5 mainly moderate nuclear staining. In addition 6 tumors presented moderate to strong cytoplasmic expression of the protein, considered abnormal. Considering tumors with weak or negative expression of BRCA1, 40% presents BRCA1 or BRCA2 mutations. The relevance of the absence of BRCA1 expression, or function impairment of BRCA1 and/or BRCA2 due to somatic mutations, relays on the possible treatment with PARP1 inhibitors. Supported by FONDECYT 1120200.

Citation Format: Carolina Alvarez, David Wiener, Patricia Gajardo, Wanda Fernandez, Valeria Cornejo, Jorge Gamboa, Pilar Carvallo. Identification of BRCA1 and BRCA2 somatic mutations in breast cancer tumors with loss of BRCA1 nuclear expression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1554. doi:10.1158/1538-7445.AM2014-1554
Abstract 1555: TP53 oncomorphic mutations are associated with resistance to platinum- and taxol-based standard chemotherapy in advanced serous ovarian cancer patients
Authors: Brachova Pavla , Dai Donghai , Carlson Mathew , Goodheart Michael , Thiel Kristina , Devor Eric , Leslie Kimberly
Published: May 2015

Mutations in the tumor suppressor gene TP53 abrogate the tumor suppressive functions of the p53 protein, and some mutations endow the mutant protein with oncogenic activity. These specific mutations we term oncomorphic. Many studies have attempted to correlate p53 mutation status with patient outcomes; however the results have been controversial due to indiscriminate categorization of all TP53 mutations into one group. Using advanced ovarian cancer as a model, our objective was to determine the relationship of oncomorphic TP53 mutations with the development of chemoresistance in advanced serous ovarian cancer patients uniformly treated with platinum and taxol primary chemotherapy. Clinical, genetic, and protein expression data from 264 high-grade serous ovarian cancer patients were downloaded from The Cancer Genome Atlas (TCGA) data portal. Associations between mutation types, chemoresistance, and progression-free survival (PFS) were calculated for patients harboring oncomorphic TP53 mutations, loss of function mutations, or unclassified mutations. Using a large and uniform patient population, we demonstrate the phenomenon of oncomorphic mutations in ovarian cancer. Specifically, patients with oncomorphic TP53 mutations showed significantly worse PFS when compared with patients with unclassified TP53 mutations (Log-rank, p < 0.05). Patients with oncomorphic TP53 mutations displayed higher rates of platinum resistance (X2, p = 0.0024) and an almost 60% higher chance of recurrence (HR=1.59, 95% confidence intervals 1.09, 2.33, p = 0.016). Furthermore, tumors containing oncomorphic TP53 mutations displayed a unique protein expression profile distinct from tumors with LOF or unclassified mutations. Taken together, these data reveal that particular oncomorphic TP53 mutations are highly associated with worse patient outcomes. Importantly, not all TP53 mutations are equal, and future studies should segregate mutations based on functional consequence. These data may have important implications for the future treatment of patients based on which TP53 mutation is present.

Citation Format: Pavla Brachova, Donghai Dai, Mathew Carlson, Michael Goodheart, Kristina Thiel, Eric Devor, Kimberly Leslie. TP53 oncomorphic mutations are associated with resistance to platinum- and taxol-based standard chemotherapy in advanced serous ovarian cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1555. doi:10.1158/1538-7445.AM2014-1555
Abstract 1560: Loss-of-function JAK1 mutations reveal a new role of protein tyrosine kinase mutations in human cancer
Authors: Ren Yuan , Zhang Yonghong , Liu Richard Z. , Fenstermacher David A. , Wright Kenneth L. , Teer Jamie K. , Wu Jie
Published: May 2015

Protein tyrosine kinases (PTKs) have been perceived mostly as proto-oncogenes. PTK mutations that have been studied in human cancer usually have increased kinase activities to drive malignant phenotypes. Thus, these PTK mutations are gain-of-function (GOF) mutations. By analyzing the targeted exome sequencing data of 3,274 human tumors from 48 different tissues in the Total Cancer Care (TCC) tumor bank, we have found JAK1 truncating mutations in 36 of 635 gynecologic tumors. The highest JAK1 truncating mutation rate (9.5%) was found in endometrial cancer. These truncating mutations result in the loss of the JAK1 PTK domain. Thus, they are loss-of-function (LOF) mutations. JAK1 truncating mutations in the Cancer Cell Line Encyclopedia (CCLE) databank also occur most often in endometrial cancer cell lines. Analysis of mutation sites identified three mutation hot spots. Re-sequencing of cancer cell lines, primary tumors, and matched normal tissues confirmed JAK1 mutations and revealed that these JAK1 mutations are somatic. JAK1 and JAK2 mediate interferon (IFN)-γ-regulated tumor immune surveillance. Recurrent JAK2 LOF mutations were also found in TCC tumors but at lower rates. Similar to JAK1, the highest rate of LOF JAK2 mutations was found in endometrial/uterine cancer. No JAK1 or JAK2 truncating mutation was identified in 130 samples from hematological malignancies. Functional assays show that JAK1-deficient cancer cells are defective in IFN-γ-induced expression of tumor antigen processing machinery proteins LMP2 and TAP1 and cell surface expression of HLA molecules. These data identify recurrent JAK1 deficiency in endometrial cancer that could impair tumor antigen presentation by the MHC class I complex to cytotoxic T lymphocytes. Our findings suggest that JAK1 is a PTK tumor suppressor and reveal a novel mechanism of tumor immune evasion via LOF mutations in a PTK.

Citation Format: Yuan Ren, Yonghong Zhang, Richard Z. Liu, David A. Fenstermacher, Kenneth L. Wright, Jamie K. Teer, Jie Wu. Loss-of-function JAK1 mutations reveal a new role of protein tyrosine kinase mutations in human cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1560. doi:10.1158/1538-7445.AM2014-1560
Abstract 1564: Spanxa1 is a novel mediator of epithelial-mesenchymal transition in lung cancer
Authors: Hsu Shih-Chun , Hsiao Yi-Jing , Yu Sing-Liang , Lee Ming-Shyue
Published: May 2015

SPANX (Sperm protein associated with the nucleus, X-linked) gene family express throughout spermatogenesis in agreement with a potential role in sperm development. It has been reported as a CTA (Cancer/Testis Antigen), which was highly expressed in testis and several cancers, such as gliobastoma, melanoma, testicular germ tumor and hematologic malignancies. Nevertheless, the function and molecular mechanism of SPANX involved in the cancer progression are greatly unknown. We found that the SPANX gene family member A1 (SPANXA1) was high-expressed in CL1-0, a less metastatic lung cancer cell line, and low-expressed in CL1-5, a more metastatic cell line. In vitro assays showed that the enforced expression of SPANXA1 suppressed the abilities of migration and invasion, and induced a dramatically mesenchymal - epithelial transition (EMT). In contrast, knock-down SPANXA1 could increase the cell mobility and invasiveness. To identify the underlying mechanism of SPANXA1, RNAs derived from SPANXA1-expressed cells and mock controls wrer assayed by gene expression microarrays and pathway analysis. The results showed that 1024 genes were differentially expressed in presence of SPANXA1 and six of top ten pathways belonged to EMT-related pathways analyzed by MetaCore software. The fact implied that SPANXA1 largely induces the EMT-related signalings. Quantitative RT-PCR assays were used to validate the altered expression of certain important genes induced by SPANXA1 in the identified pathways. The EMT regulating transcription factor, Slug, was decreased in SPAXA1-overexpressed cells and it also acts as a repressor for E-cadherin expression. We found that SPANXA1 mainly localized at nucleus and inhibited the formation of filopodia assayed by immunofluoresence staining. To explore the function of SPANXA1 in the nucleus, we performed Chromatin IP sequencing and the results elucidated that SPANXA1 might bind onto chromosome and regulate gene expressions. Furthermore, mouse model demonstrated that the distal metastasis was dramatically decreased in SPANXA1-overexpressed cells compared with the mock controls. These data suggest that SPANXA1 is a novel EMT modulator involved in lung cancer progression and implies the potential therapeutic role of SPANXA1 in the future.

Citation Format: Shih-Chun Hsu, Yi-Jing Hsiao, Sing-Liang Yu, Ming-Shyue Lee. Spanxa1 is a novel mediator of epithelial-mesenchymal transition in lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1564. doi:10.1158/1538-7445.AM2014-1564
Abstract 1585: Chronic induction of breast cell carcinogenesis by multiple environmental and dietary carcinogens
Authors: Pluchino Lenora A. , Wang Hwa-Chain R.
Published: May 2015

Breast cancer, the most common type of cancer among North American

and European women, is mostly sporadic and attributable to long-term exposure

to small quantities of multiple carcinogens.

To understand how multiple carcinogens act together to induce breast

cell carcinogenesis, we investigated the activity of the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone

(NNK), the environmental carcinogen benzo[a]pyrene (B[a]P), and the dietary

carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Non-cancerous human breast epithelial MCF10A

cells were exposed to NNK, B[a]P, and PhIP sequentially or in combination and

then analyzed for the acquisition of cancerous properties/endpoints. Transient endpoints included DNA damage, Ras-Erk-Nox pathway

activation, reactive oxygen species (ROS) elevation,

and increased cell proliferation. Constitutive endpoints

included reduced dependence on growth factors,

anchorage-independent growth, Ras-Erk-Nox pathway activation, and increased ROS

and cell proliferation. To detect agents that

can intervene with cellular carcinogenesis induced by NNK, B[a]P, and PhIP, we

investigated the activity of green tea catechins effective in blocking these

cancerous endpoints. Our study revealed, for the

first time, that combined NNK and B[a]P enhanced

breast cell carcinogenesis chronically induced by PhIP. Co-exposure was more potent than sequential exposure to

induce initiation of cellular carcinogenesis measured by induction of transient

endpoints in a single exposure and progression of carcinogenesis measured by

cellular acquisition of constitutive endpoints in cumulative exposures. Our study also revealed that combined ECG and EGCG, at non-cytotoxic levels, was more effective

than individual agents in intervention of cellular carcinogenesis induced by

combined NNK, B[a]P, and PhIP. Thus, co-exposure

to NNK, B[a]P, and PhIP should be critically examined in epidemiological

studies to reveal the impact of these carcinogens in the development of

sporadic breast cancer. Use of combined

ECG and EGCG should be seriously considered for chemoprevention of breast cell

carcinogenesis associated with long-term exposure to environmental and dietary

carcinogens.

Citation Format: Lenora A. Pluchino, Hwa-Chain R. Wang. Chronic induction of breast cell carcinogenesis by multiple environmental and dietary carcinogens. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1585. doi:10.1158/1538-7445.AM2014-1585
Abstract 1587: Mechanistic role of cytochrome P4501B1 in DNA adduct formation by 3-methylcholanthrene in mice: Implications for lung cancer in humans
Authors: Moorthy Bhagavatula , Zhou Guodong , Wang Lihua , Kondraganti Sudha R. , Jiang Weiwu
Published: May 2015

Humans are frequently exposed to polycyclic aromatic hydrocarbons (PAHs). Cytochrome P450 (CYP)1A and 1B1 enzymes are known to play important roles in the activation of PAHs such as 3-methylcholanthrene (MC) to DNA-binding metabolites, which in turn mediate carcinogenesis in target organs such as lung, breast, and skin. In this investigation, we tested the hypothesis that mice lacking the gene for Cyp1b1 will show attenuation of MC-DNA adduct formation, compared to WT mice. Eight week-old female wild type (WT) (C57BL/6J) mice or mice lacking the gene for Cyp1b1 were treated with MC (100 µmol/kg), once daily for 2 days, and animals sacrificed 1 day after the last treatment. DNA adducts from liver and lung genomic DNA were analyzed by the 32P-postabeling assay. Control animals received only the vehicle corn oil (CO). In WT mice, CO-treated group did not show any adducts, whereas MC-treated animals showed 11 adduct spots in the lung. In Cyp1b1-null mice, although there were no qualitative differences in the pulmonary adduct pattern, the levels of each adduct was markedly suppressed (25-90%), with total levels of adducts being about 15% of those formed in WT mice. In liver of WT as well as Cyp1b1-null mice, MC induced formation of 11 adducts, and the adduct pattern was similar to that of lung, but the total levels of adducts in WT mice were about a third of those seen in the lungs of the corresponding animals. In Cyp1b1-null mice, MC adducts were significantly decreased compared to WT mice, but the extent of attenuation in liver was lesser than that in lung. These results suggest that CYP1B1 plays a major role in formation of DNA adducts from MC. Because DNA adducts are pre-mutagenic lesions, our results are highly relevant to PAH-mediated pulmonary carcinogenesis. Our data suggest that CYP1B1 could be a molecular target for developing preventive and therapeutic strategies against lung cancer in humans.

Citation Format: Bhagavatula Moorthy, Guodong Zhou, Lihua Wang, Sudha R. Kondraganti, Weiwu Jiang. Mechanistic role of cytochrome P4501B1 in DNA adduct formation by 3-methylcholanthrene in mice: Implications for lung cancer in humans. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1587. doi:10.1158/1538-7445.AM2014-1587
Abstract 1588: Detection in vivo of a novel endogenous etheno DNA adduct derived from arachidonic acid and the effects of antioxidants on its formation
Authors: Fu Ying , Dyba Marcin , Pan Jishen , Schultz Casey , Yang Peiying , Desai Dhimant , Amin Shantu , Chung Fung-Lung
Published: May 2015

Previous studies showed that the 7-(1′,2′-dihydroxyheptyl) substituted etheno DNA adducts are products from reactions with epoxide of (E)-4-hydroxy-2-nonenal (HNE), an oxidation product of n-6 polyunsaturated fatty acids (PUFAs). In this work, we report the detection of 7-(1′,2′-dihydroxyheptyl)-1,N6-ethenodeoxyadenosine (DHHedA) in rodent and human tissues by two independent methods: a 32P-postlabeling/HPLC method and an isotope dilution liquid chromatography electrospray ionization tandem mass spectrometry method (ID-LC-ESI-MS/MS). This study demonstrated for the first time that DHHedA is a background DNA lesion in vivo. We showed that DHHedA can be formed upon incubation of arachidonic acid (AA) with deoxyadenosine (dA), supporting the notion that n-6 PUFAs are the endogenous source of DHHedA formation. Because the cyclic adducts are derived from the oxidation of PUFAs, we subsequently examined the effects of antioxidants, α-lipoic acid, polyphenon E and vitamin E on the formation of DHHedA and γ-hydroxy-1,N2-propanodeoxyguanosine (γ-OHPdG), a widely studied acrolein-derived cyclic adduct arisen from oxidized n-3 and n-6 PUFAs, in the livers of Long Evans Cinnamon (LEC) rats. LEC rats are inflicted with elevated lipid peroxidation and prone to the development of hepatocellular carcinomas. The results showed that while the survival of LEC rats was increased significantly by dietary α-lipoic acid, none of the antioxidants inhibited the formation of DHHedA and only polyphenon E treatment decreased the formation of γ-OHPdG. In contrast, vitamin E caused an increase in the formation of both γ-OHPdG and DHHedA in the livers of LEC rats. This work was supported by the NCI grant: RO1-CA-134892.

Citation Format: Ying Fu, Marcin Dyba, Jishen Pan, Casey Schultz, Peiying Yang, Dhimant Desai, Shantu Amin, Fung-Lung Chung. Detection in vivo of a novel endogenous etheno DNA adduct derived from arachidonic acid and the effects of antioxidants on its formation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1588. doi:10.1158/1538-7445.AM2014-1588
Abstract 1948: Human papillomavirus enhances oral cancer stem cell phenotype by regulating microRNA-181
Authors: Lee Sung Hee Hee , Rigas Nicole , Lee Chang-Ryul , Han Jiho , Kim Reuben , Kang Mo , Park No-Hee , Shin Ki-Hyuk
Published: May 2015

High-risk human papillomaviruses (e.g., HPV-16 and HPV-18) are closely associated with the development of human head and neck squamous cell carcinomas (HNSCC), including malignant lesions in the oral cavity. However, less is known about the possible role and the underlying mechanisms of HPV in enhancing cancer progression and promoting the virulence of cancer, including oral squamous cell carcinoma (OSCC). Cancer stem cells (CSCs) play crucial role in cancer progression, metastasis and recurrence, and are epigenetically regulated by microRNAs (miRNAs). Recent studies demonstrated that miRNA expression is affected by HPV status in HNSCC, suggesting a possible role of HPV in epigenetic regulation of CSCs. In this study, we investigated the role of high-risk HPV on malignant and CSC phenotypes as well as epigenetic regulation in OSCC. Incorporation of HPV-16 whole genome into HPV-negative OSCCs resulted in enhancement of malignant phenotypes (e.g., increased anchorage independent growth, proliferation in organotypic epithelial raft culture, and tumorigenicity in nude mice) and CSC phenotypes (e.g., increased CSC-related markers, self-renewal, and migration/invasion). High-throughput miRNA expression analysis revealed that miRNA-181 family members were significantly underexpressed in the HPV16-harboring OSCCs. HPV-16 downregulated the promoter activity of miR-181a. Furthermore, restoration of miR-181 in the presence of HPV-16 suppressed CSC-like phenotype. Taken together our data indicate that high-risk HPV enhances stemness phenotypes in OSCC by epigenetic regulation of miR-181 expression.

Citation Format: Sung Hee Hee Lee, Nicole Rigas, Chang-Ryul Lee, Jiho Han, Reuben Kim, Mo Kang, No-Hee Park, Ki-Hyuk Shin. Human papillomavirus enhances oral cancer stem cell phenotype by regulating microRNA-181. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1948. doi:10.1158/1538-7445.AM2014-1948
Abstract 1950: SMURF1 inhibition reduces cancer stem cell-like population in head and neck squamous cell carcinoma
Authors: Khammanivong Ali , Gopalakrishnan Raj , Dickerson Erin B.
Published: May 2015

Cancer stem cells (CSCs) possess capabilities for long-term survival (quiescence), self-renewal, and tumor initiation thought to be responsible for drug resistance and treatment failures for many malignancies, including head and neck squamous cell carcinoma (HNSCC). Previously, we showed that the activity of bone morphogenetic protein (BMP), a key regulator of cell differentiation, proliferation, migration, and apoptosis, is inhibited in CSC-like populations enriched from HNSCC cell lines. In contrast, the BMP signaling inhibitor SMAD specific E3 ubiquitin protein ligase 1 (SMURF1) and CD44, a marker of CSCs, were upregulated.

Objectives: In this study, we characterized the role of SMURF1 for CSC maintenance and differentiating growth and identified a population of cells regulated by SMURF1.

Methods: We generated a stable knockdown of SMURF1 expression in CSC-like populations using a lentivirus delivered shRNA interference construct. Long-term colony formation and Calcein AM dye efflux assays were performed to assess CSC maintenance and differentiating growth.

Results: Knockdown of SMURF1 expression by shRNA reduced viability and colony formation of CSC-like cells grown in three-dimensional culture. Furthermore, reduced SMURF1 expression diminished the number of cells capable of dye efflux and decreased CD44 expression (CD44-high) in a select cell population.

Conclusion: We interpret our data to suggest that SMURF1 inhibition of BMP signaling potentiates the long-term survival of HNSCC CSCs. Reduced dye efflux and expression of CD44-high cells suggests decreased maintenance of CSC-like populations along with an increased potential for differentiating growth. Targeting SMURF1 may provide a long-term benefit for HNSCC treatment to reduce resistant CSC populations and disease recurrence.

Citation Format: Ali Khammanivong, Raj Gopalakrishnan, Erin B. Dickerson. SMURF1 inhibition reduces cancer stem cell-like population in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1950. doi:10.1158/1538-7445.AM2014-1950
Abstract 1951: Role of embryonic signaling pathways in lung cancer
Authors: Singh Samer , Nguyen Dao M. , Capobianco Anthony J. , Robbins David J.
Published: May 2015

Background: Embryonic signaling pathways that play an important role during embryo development, later on support the maintenance of adult stem cells and are also found activated in various cancers. The activated embryonic signaling, mainly Hedgehog (HH), WNT and NOTCH, have been shown to be activated in a number of cancers and demonstrated to promote growth, provide survival advantage and resistance to various drugs. The role of these embryonic signaling pathways in the development of lung cancer is not well defined. Identification of particular pathways active in cancer and elucidation of their mechanism of action in cancer development could provide a basis of devising a successful therapeutic intervention.

Objective: Define the role of embryonic signaling pathways in cancer cells.

Methodology: The presence and functional interaction of various embryonic signaling pathways in lung cancer was evaluated using human non-small cell lung carcinoma cell lines (e.g. HOP62, A549, H23, H522) as model system. The signaling pathways were inhibited using specific small molecule inhibitors and shRNAs mediated knockdown of the pathway components. The lentiviruses expressing shRNA were made and used to deliver the shRNA in NSCLC cells. The effect on the target gene expression (quantitative real time RT-PCR and western blotting), cell proliferation (ATP quantitation), cell migration (scratch/wound assay) and anchorage independent growth (soft agar assay), was evaluated. Currently, the activation of pathways in cancer cells of primary lung cancer samples is being evaluated by immunohistochemistry (IHC) and RNA in situ hybridization (ISH).

Preliminary Results and Conclusions: Attenuation of individual embryonic signaling pathways using specific small molecule inhibitors and shRNAs, indicated crosstalk and hierarchy of these pathways in controlling various processes in tested cancer cells. Our results suggest the predominance of HH signaling in non-small cell lung carcinoma. We are in the process of expanding our observation to more cell lines and human cancer samples. Our work will establish the role of embryonic signaling pathways in lung cancer and would further help in establishing a framework for future therapeutic intervention.

Citation Format: Samer Singh, Dao M. Nguyen, Anthony J. Capobianco, David J. Robbins. Role of embryonic signaling pathways in lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1951. doi:10.1158/1538-7445.AM2014-1951
Abstract 1954: RNA-binding protein Musashi1 activates STAT3 signaling via PKR phosphorylation and promotes tumorigenicity of glioblastoma cells
Authors: Chen Hsiao-yun , Chiou Shih-Hwa
Published: May 2015

Glioblastoma multiforme (GBM) is one of the deadly malignant brain tumors for human. GBM is easily recurrent and resistant to the clinical approaches. Those clinical interventions are considered a minor stress, therefore, promotes tumorigenicity of cancer cells. RNA-binding protein, Musashi-1 (Msi-1), is highly expressed in the central nervous system and it is known to act a vital role in regulation of neuron cell development. Moreover, Msi-1 is elevated in several cancer tissues and confirmed as a positive regulator in cancer progression. In this present article, we hypothesized that Msi-1 promotes cell tumorigenicity and enhances drug resistance under expose to stress condition in human GBM cells. We validated over expression of Msi-1 lead a protective effect in mitigating drug-induced cells death. Under stress stimulation, overexpression of Msi-1 triggers Msi-1-granules generation, stress granules (SGs) markers such as p-PKR, p-PERK and p-eIF2-α phosphorylation. The activated-PKR contributes down-stream leading to enhanced STAT3 activity by increased tyrosine phosphorylation of STAT3, thereby enhancing tumorigenicity in GBM cells. Knocking down of Msi-1 and PKR obviously antagonist those finding, inhibitors of PKR and STAT3 reduced cells viability under expose to stress condition. Our findings concluded that Msi-1 contributes a positive role in regulation of tumorigenicity and drug resistance to GBM cells under stress condition by modulating PKR/STAT3 mechanism.

Citation Format: Hsiao-yun Chen, Shih-Hwa Chiou. RNA-binding protein Musashi1 activates STAT3 signaling via PKR phosphorylation and promotes tumorigenicity of glioblastoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1954. doi:10.1158/1538-7445.AM2014-1954
Abstract 196: Targeting breast cancer stem cells with a panel of potential ALDH1A3 inhibitors
Authors: Thomas Margaret L. , Wallace Melissa , Giacomantonio Carman , Marcato Paola
Published: May 2015

Aldehyde dehydrogenase (ALDH) isoform ALDH1A3 is selectively expressed in breast cancer stem cells (CSCs) and is a key mediator in breast cancer progression via initiation of retinoic acid (RA) signaling and expression of RA-inducible genes. Expression of ALDH1A3 and its induced RA-inducible genes are associated with less favorable patient outcomes. This suggests that ALDH1A3 represents a novel anti-breast cancer therapeutic target. To target the specific ALDH1A3 isoform, a panel of general ALDH inhibitors was attained and the compounds’ effectiveness in targeting ALDH1A3 activity was quantified by their ability to decrease expression of the downstream genes retinoic acid receptor beta (RARβ) and retinoic acid receptor responder protein 1 (RARRES1). Eleven compounds were tested in total, and the essential oil citral was shown to significantly decrease ALDH1A3-mediated expression of both RA-inducible genes in a dose dependent manner. Citral is already used in cosmetics and food additives and recent work shows that it can induce apoptosis of breast cancer cells making it a promising therapeutic agent and a potential subject of a future xenograft study in mice to determine its effects on CSCs in vivo.

Citation Format: Margaret L. Thomas, Melissa Wallace, Carman Giacomantonio, Paola Marcato. Targeting breast cancer stem cells with a panel of potential ALDH1A3 inhibitors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 196. doi:10.1158/1538-7445.AM2014-196
Abstract 1974: Ovulatory wound: the site of origin for ovarian serous carcinoma
Authors: Yang-Hartwich Yang , Gurrea Marta , Sumi Natalia , Holmberg Jennie , Craveiro Vinicius , Alvero Ayesha , Mor Gil
Published: May 2015

Background: New clinical and molecular studies suggest that HGSOC originates from the neighboring fallopian tubes, rather than the surface epithelial layer of the ovary. However, very little is known about the process of tubal malignant cells migrating and implanting into ovary. Our research focuses on understanding how the extraovarian malignant cells implant into the ovary and how ovulation affects the process of their migration and implantation.

Method: Tumor initiating cells (TICs) isolated from human HGSOC were labeled by red fluorescent protein (RFP)-expressing lentivirus and subcutaneously or intrauterine injected to immunocompromised mice. Hormones (PMS and HCG) were interperitoneally injected to induce superovulation in mice. In the in vitro models, TICs were cultured with the granulosa cell conditional medium and the extra cellular matrix (ECM) extracted from ovarian stroma cells to evaluate their effects on the migration and attachment of TICs

Result: Superovulation increased the ovarian tumor-forming rate from 38% (15/39 in control group) to 100% (24/24 in supervolation group). The increased frequency of ovulation enhances the formation of ovarian tumors in mouse models. In the intrauterine injection model, we were able to trace the migration of TICs towards ovaries and capture the early implantation of TICs inside ovaries in 5-10 days after the injection. We detected stage I ovarian tumors in mice. The tumors are encapsulated in the ovary and covered by the intact surface epithelial layer. These tumor cells closely interacted with the stroma of ovary, particularly the structure of corpus luteum. The granulosa cells secreted stromal cell-derived factor 1 (SDF-1, also known as CXCL12). SDF-1 attracts TICs that express CXCR4 (the receptor of SDF-1) to ovulatory wounds. Upon ovulation the ovulatory wounds expose the stroma of ovary, which provides a collagen enriched ECM for TICs to attach.

Conclusion: The unique ovulatory wound microenvironment attracts the malignant cells to migrate towards the ovary and creates access for the malignant cells to implant in the collagen-enriched ECM in the stroma of ovary. The new mechanisms revealed by our data may explain why increased ovulation is associated with epithelial ovarian cancer. Our findings support the “extra-ovarian origin” theory and will help us better understand the initiation and progression of HGSOC.

Citation Format: Yang Yang-Hartwich, Marta Gurrea, Natalia Sumi, Jennie Holmberg, Vinicius Craveiro, Ayesha Alvero, Gil Mor. Ovulatory wound: the site of origin for ovarian serous carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1974. doi:10.1158/1538-7445.AM2014-1974
Abstract 1976: MDR expression/ activity may serve as potential biomarker in developing therapeutic drugs for AML therapy
Authors: Rudra-Ganguly Nandini , Lowe Christine , Shiwalkar Mukta , Guevara Claudia I. , Kemball Christopher , Wu Min Michelle , Virata Cyrus , Verlinsky Alla , Hsu Ssucheng J , Mattie Michael , Yeh William , Yang Peng , Moon Sung-Ju , Joseph Ingrid , Stover David R. , Pereira Daniel S. , Jacksosn Dowdy
Published: May 2015

Multidrug resistance may play a critical role in developing cancer therapeutics for acute leukemia. The expression of the ATP binding cassette (ABC) transporter Pgp/MDR1 (also known as ABCB1) has been reported in AML patients and correlates with poor clinical outcome. Additional transporters like MRP1 and BCRP also appeared to contribute towards this resistance mechanism. We have profiled a panel of AML tumor cell lines and tumor xenografts for both expression and activity of Pgp/MDR1 via FACS and MSD;. Our profiling also includes analysis of the known SNPs associated with these transporters in key models. We have evaluated the sensitivity of the AML models to a series of standard of care therapies, including auristatins, in both in vitro and in vivo studies. These payloads alone or when conjugated to antibodies as antibody drug conjugates (ADCs) are known substrates of the ABC transporters, Using Pgp inhibitors, we could restore the sensitivity of the MDR expressing cells to the cytotoxic drugs and ADCs, which confirmed that the drugs were MDR substrates. These inhibitors could also block the activity of the Pgp pump. We have also observed limited activity in vivo with these ADCs when used in a MDR positive model. Our initial observation indicates that a threshold level of activity of MDR1 may be critical to confer resistance to these standard of care molecules and antibody drug conjugates using similar payloads. Currently, we are investigating the contribution of other efflux pumps in this mechanism of drug resistance MRP1 and BCRP, in similar manner. This can also aid in identifying cytotoxic drugs that are able to bypass the resistance mediated by these efflux pumps. Additional work is underway to characterize patient samples and identify MDR threshold level of activity as a potential predictive biomarker in developing AML therapeutics.

Citation Format: Nandini Rudra-Ganguly, Christine Lowe, Mukta Shiwalkar, Claudia I. Guevara, Christopher Kemball, Min Michelle Wu, Cyrus Virata, Alla Verlinsky, Ssucheng J Hsu, Michael Mattie, William Yeh, Peng Yang, Sung-Ju Moon, Ingrid Joseph, David R. Stover, Daniel S. Pereira, Dowdy Jacksosn. MDR expression/ activity may serve as potential biomarker in developing therapeutic drugs for AML therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1976. doi:10.1158/1538-7445.AM2014-1976
Abstract 1979: Three-dimensional Gelfoam® histoculture enables cancer cells to mimic in vivo cancer cell cycling as visualized with FUCCI imaging
Authors: Yano Shuya , Miwa Shinji , Mii Sumiyuki , Hiroshima Yukihiko , Uehara Fuminari , Kishimoto Hiroyuki , Tazawa Hiroshi , Zhao Ming , Fujiwara Toshiyoshi , Hoffman Robert M.
Published: May 2015

Collagen sponge-gel histoculture was developed by Leighton (In: Encyclopedia of Life Sciences. John Wiley and Sons, Ltd.: Chichester, 2010, Published Online. DOI: 10.1002/9780470015902.a0002573.pub2). In the 1950s, Leighton showed that placing cells in histoculture enables them to form 3-dimensional structures. Because of its architectural resemblance to native tissue, sponge gel histoculture represents a unique in vivo-like model to study cancer-cell behavior (Brenner's Encyclopedia of Genetics, 2nd Ed., Vol. 7, pp. 73-76. Elsevier, 2013). For example, Leighton observed that when C3HBA mouse mammary adenocarcinoma cells were grown on sponge-matrix histoculture, the cells aggregated similar to the original in vitro tumor. Distinct structures were formed within the tumors such as lumina and stromal elements, with some of the glandular structures similar to the original tumor (above-reference). We have further developed sponge gel histoculture using Gelfoam to grow tumors, nerves, hair follicles, skin with growing hair (above-reference). In the present report, we use fluorescence ubiquitination cell cycle indicator (FUCCI) imaging and Gelfoam® collagen sponge gel histoculture to demonstrate that the cell cycle phase distribution of cancer cells in Gelfoam® and in vivo tumors is similar whereby only the surface cells proliferate and interior cells are quiescent in G1/G0. This is markedly contrary to 2D culture where most cancer cells cycle. This observation explains, at least in part, the resistance of solid tumors to chemotherapy which targets only cycling cells.

Citation Format: Shuya Yano, Shinji Miwa, Sumiyuki Mii, Yukihiko Hiroshima, Fuminari Uehara, Hiroyuki Kishimoto, Hiroshi Tazawa, Ming Zhao, Toshiyoshi Fujiwara, Robert M. Hoffman. Three-dimensional Gelfoam® histoculture enables cancer cells to mimic in vivo cancer cell cycling as visualized with FUCCI imaging. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1979. doi:10.1158/1538-7445.AM2014-1979