bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1271: Alcohol intake and breast cancer in the European Prospective investigation into Cancer and Nutrition (EPIC) study
Authors: Romieu Isabelle , Scoccianti Chiara , Chajes Veronique , Batlle Jordi De , Rinaldi Sabina
Published: May 2015

Context: Alcohol intake has been positively associated to breast cancer among both pre and postmenopausal women; however results are inconclusive with regards to tumor hormonal receptor status, and potential modifying factors such as age at start drinking, and dietary fiber.

Objective: To investigate the relation between alcohol intake and the risk of breast cancer using data from the European Prospective Investigation into Cancer and Nutrition (EPIC).

Design, Setting, and Participants: Prospective observational study of 334,850 women mostly aged 35-70 years at baseline enrolled in the ten countries of the EPIC study and followed up for 11.0 years on average. Information on alcohol intake at baseline and average lifetime alcohol intake were calculated from country-specific dietary and lifestyle questionnaires.

Main Outcome Measures: Hazard ratios (HR) of developing invasive breast cancer according to hormonal receptor status.

Results: During 3,670,439 person-years, 11,576 incident breast cancer cases were diagnosed. Increasing alcohol intake was significantly related to breast cancer risk, for each 10g/day increase in alcohol intake the HR increased by 4.2% (95% CI, 2.7% -5.8%). Taking 0 to 5g/day as reference, alcohol intake of >5 to 15 g/day was related to a 5.9% increase of breast cancer risk (95% CI, 1% -11%). A significant increasing trend was observed between alcohol intake for ER+/PR+, ER-/PR-, HER2- and ER-/PR-/HER2-tumors. The risk of breast cancer appeared stronger among women who started drinking prior to first full-time pregnancy and among drinkers (>15g/day) with low intake of fiber.

Conclusions: Our results confirm the association between alcohol intake and breast cancer risk, and suggest that some other risk factors such as fiber intake, may modulate this association. Women should be advised to control their alcohol intake, and increase their intake of fiber.

Citation Format: Isabelle Romieu, Chiara Scoccianti, Veronique Chajes, Jordi De Batlle, Sabina Rinaldi. Alcohol intake and breast cancer in the European Prospective investigation into Cancer and Nutrition (EPIC) study. [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 1271. doi:10.1158/1538-7445.AM2014-1271
Abstract 1280: Germline PALB2 genetic variations of familial esophageal squamous cell carcinoma (ESCC) in Henan, a high risk ESCC region
Authors: Ko Josephine Mun Yee , Wang Li Dong , Lung Maria
Published: May 2015

Background and aims:

ESCC is often a deadly cancer diagnosed at late stage with a 5-year survival rate less than 10% in advanced cancers. It is important to elucidate the molecular genetic basis for this deadly cancer to achieve the ultimate goal of early cancer detection and improved clinical management. Our aim is to understand the genetic basis of inherited ESCC by deciphering PALB2 mutation status in high risk northern China. By investigation of PALB2 germline mutations and variants, as compared to healthy individuals, the genetic basis and genomic risk factor associations with inherited EC will be clarified.

Methods:

Blood was collected from high-risk Henan familial history-positive (FH+) individuals. Blood DNAs were extracted for Sanger sequencing analysis.

Results:

By Sanger sequencing with primers covering more than 90% of the PALB2 coding sequence, PALB2 variants were detected in exon 4 for 4% (2/50) FH+ ESCC patients from Henan, but no germline protein truncation variations were observed. Since mutations were only observed in exon 4, further mutation screening were confined to exon 4 of PALB2 with an additional 300 FH+ Henan ESCC, 283 FH- Henan ESCC, and 364 Henan non-ESCC control individuals. However, no mutations were detected in the validation samples.

Conclusions:

Infrequent PALB2 germline mutations were detected in Henan FH+ ESCC patients, suggesting PALB2 may be involved in a small proportion of familial ESCC in Henan. Further study is necessary to clarify if the identified mutations are pathogenic.

Citation Format: Josephine Mun Yee Ko, Li Dong Wang, Maria Lung. Germline PALB2 genetic variations of familial esophageal squamous cell carcinoma (ESCC) in Henan, a high risk ESCC region. [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 1280. doi:10.1158/1538-7445.AM2014-1280
Abstract 1289: Breast cancer predisposition in multiple endocrine neoplasia type 1
Authors: Dreijerink Koen , Conemans Elfi , Cornelissen Chantal , Blokland Marja Van , Mannstadt Michael , Pieterman Carla , Herder Wouter D. , Havekes Bas , Fliers Eric , Hermus Ad , Vriens Menno , Timmers Marc , Wall Elsken Van der , Brown Myles , Luijt Rob Van der , Diest Paul van , Valk Gerlof
Published: May 2015

Recent genetic and animal studies have suggested that the Multiple Endocrine Neoplasia type 1 (MEN1) gene is involved in breast carcinogenesis. In addition, the MEN1 gene product menin is a regulator of estrogen receptor alpha function in vitro.

In this retrospective study in two independent cohorts we aimed to investigate the prevalence of breast cancer in female MEN1 patients and to characterize MEN1-related breast tumors.

The Dutch MEN1 cohort contains data from 190 female MEN1 patients from 1990-2011. The second cohort from the U.S. has data from 68 female patients from 2004-2013. Observations were matched with demographic and cancer incidence and prevalence information from the general Dutch and U.S. populations. Breast cancer tissues from MEN1 patients were analyzed immunohistochemically and loss of heterozygosity (LOH) at the MEN1 gene locus was assessed.

Females with MEN1 were found to be at increased risk to develop breast cancer (relative risks 2.83 and 3.20). Eighty percent of MEN1-related breast tumors showed loss of expression of menin and in a subset also loss of heterozygosity at the MEN1 gene locus was found.

MEN1 is a breast cancer susceptibility syndrome. Further research will determine the relevance of the MEN1 gene for patients with non-familial breast cancer. Intensified breast cancer screening should be considered for female MEN1 patients.

Citation Format: Koen Dreijerink, Elfi Conemans, Chantal Cornelissen, Marja Van Blokland, Michael Mannstadt, Carla Pieterman, Wouter De Herder, Bas Havekes, Eric Fliers, Ad Hermus, Menno Vriens, Marc Timmers, Elsken Van der Wall, Myles Brown, Rob Van der Luijt, Paul van Diest, Gerlof Valk. Breast cancer predisposition in multiple endocrine neoplasia type 1. [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 1289. doi:10.1158/1538-7445.AM2014-1289
Abstract 1290: Survival in familial breast cancer: Novel findings based on a nationwide prospective cohort study
Authors: Kharazmi Elham , Försti Asta , Sundquist Kristina , Hemminki Kari
Published: May 2015

Objective: To find out whether breast cancer (BC) patients with positive family history of concordant cancer are diagnosed at earlier TNM stages and to explore the survival in familial breast cancer compared to sporadic cases who were diagnosed at identical TNM status and staging.

Design: Nationwide prospective cohort study.

Setting and method: The nationwide Swedish Family-Cancer Database including all Swedes born after 1931 and their biological parents, totaling >14.7 million individuals. Hazard ratios (HR) were calculated for 4,709 women with BC in a first-degree relative (FDR) versus 25,366 BC women without such a family history.

Results: Familial BC cases were not diagnosed at earlier stages compared to sporadic cases. There was no difference in survival of familial BC patients who were diagnosed at high TNM staging (T2-T4, N1-N3, M1) compared to sporadic cases diagnosed at the same late stage. Young BC patients (age<40) in early stages (T0-T1, N0, M0, Stage 0-I) had the worst survival when their FDR were diagnosed with single (HR: 2-3.7) or multiple (HR: 2.4-7.1) BC at any age.

Conclusion: Young BC patients (age<40), diagnosed at early stages (T0-T1, N0, M0, stage 0-I), whose FDR were diagnosed with BC at any age had the higher risk of mortality compared to sporadic cases at the same stages. Our results offer novel information for personalized genetic counseling and treatment modalities and urge the need for identifying the underling genetic component for such a difference in survival of familial breast cancer.

Citation Format: Elham Kharazmi, Asta Försti, Kristina Sundquist, Kari Hemminki. Survival in familial breast cancer: Novel findings based on a nationwide prospective cohort study. [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 1290. doi:10.1158/1538-7445.AM2014-1290
Abstract 1294: A pilot study of FBRSL1 and melanoma: What's the connection
Authors: Lilyquist Jenna Marie , Lee Pedro , Berwick Marianne , Reinberg Danny
Published: May 2015

FBRSL1 is a novel protein and a member of a heterogeneous group of proteins that form polycomb repressive complex 1 (PRC1). The canonical function of PRC1 is monoubiquitination of H2A at lysine 119 which has been associated with nucleotide excision repair of UV-induced DNA lesions; however, the role FBRSL1 plays in the PRC1 complex is unknown. Additionally, single nucleotide polymorphisms residing in FBRSL1 have been shown to modify melanoma risk following indoor tanning. Since very little is known about the function of FBRSL1, the connection between FBRSL1 and melanoma is unclear. To investigate FBRSL1 expression in relationship to melanoma, we performed an immunohistochemistry (IHC) pilot study, using antibodies against both the long and short forms of murine FBRSL1, on a small set of melanoma tumor and benign patient tissues. Our results indicate that both murine antibodies recognize FBRSL1 in human skin. The FBRSL1 amino acid sequence is highly conserved between mice and humans, but only the long isoform has been validated in humans indicating that further characterization of the human isoforms of FBRSL1 is needed. Additionally, we found FBRSL1 expression only in melanoma tissues, and not in benign tissues. Future directions include validating these results in a larger group of patients using both melanoma and benign tissues to confirm the relationship between FBRSL1 expression and melanoma. Ultimately, we will investigate the association between FBRSL1 and patient characteristics that modify melanoma risk and prognosis. We expect that these results will inform experiments to elucidate the role of FBRSL1 in melanoma.

Citation Format: Jenna Marie Lilyquist, Pedro Lee, Marianne Berwick, Danny Reinberg. A pilot study of FBRSL1 and melanoma: What's the connection. [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 1294. doi:10.1158/1538-7445.AM2014-1294
Abstract 130: Deadly KISS, Kisspeptin10 interaction with osteoblasts and breast cancer metastastic cells
Authors: Leahey Kaitlyn E. , Welch Danny R. , Chen Yu-Chi , Bussard Karen M. , Mastro Andrea M.
Published: May 2015

Current cancer research focuses on blockading metastasis with the hope of increasing patient survival and prognoses. This research has spurred an interest in metastasis suppressor genes, such as KISS-1, whose role in breast cancer remains controversial. This study focuses on the interactions among one of the proteolytic processing products of the Kiss1 protein (kisspeptin10, KP10), its receptor, KISS1R (GPR54, AXOR12), osteoblasts (bone forming cells), and bone-metastatic breast cancer cells, MDA-MB-231. Previous research suggests that treatment with KP10 reduces the ability of the cancer cells to invade a reconstituted basement membrane (Matrigel) and to migrate in response to osteosarcoma cells. The aim of this present study was to determine if KP10 affected the interaction of MC3T3-E1 osteoblasts and bone-metastatic breast cancer cells.

Our laboratory had previously determined that osteoblasts exhibit an inflammatory response when treated with conditioned medium from breast cancer cells. This inflammatory response may contribute to cancer cell proliferation in the bone. This response has not yet been characterized in the presence of KP10. I hypothesized that KP10 would down regulate the inflammatory response of osteoblasts to breast cancer cells. MDA-MB-231 breast cancer cells express theKISS1R; therefore, these cells were treated with KP10 (10-100 nM, 2hr), the medium was collected, and the osteoblasts were treated with it. KP10 significantly decreased the inflammatory response of osteoblasts to breast cancer conditioned media indicated by decreased levels of IL-6 as determined by ELISA. This research has important implications because it provides insight into how KP10 may be acting on either the breast cancer cells or the bone cells. In my future research, I will continue to characterize how KP10 can be used to prevent proliferation or activation of breast cancer metastases in the bone.

This work was supported by an undergraduate research grant from the Eberly College of Science of Penn State University.

Citation Format: Kaitlyn E. Leahey, Danny R. Welch, Yu-Chi Chen, Karen M. Bussard, Andrea M. Mastro. Deadly KISS, Kisspeptin10 interaction with osteoblasts and breast cancer metastastic 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 130. doi:10.1158/1538-7445.AM2014-130
Abstract 1300: Exploration of rare variants from exome sequencing in families with Waldenstrom macroglobulinemia (WM)
Authors: McMaster Mary L. , Goldin Lynn R. , Rotunno Melissa , He Ji , Burdette Laurie , Hutchinson Amy , Boland Joseph , Yeager Meredith , Tucker Margaret A. , Chanock Stephen J. , Caporaso Neil E.
Published: May 2015

In spite of progress in the identification of the somatic events associated with Waldenström macroglobulinemia (WM), the genetic determinants of WM susceptibility have not been identified. We have conducted exome sequencing in 32 individuals from 9 well-characterized families at high risk for WM. We sequenced 3 or more patients or obligate carriers from each family. We used Nimblegen v2.0 and v3.0 for exome capture followed by sequencing on the Illumina HiSeq2000. Among quality control measures, we required 80% of coding sequences to achieve 15x coverage. To eliminate other possible sequencing artifacts, variants found in more than 1% of samples sequenced in our laboratory from other studies were excluded. Novoalign v.2.07.14 was used for alignment and GATK was used for local re-alignment and variant calling. A large number of rare (<1% frequency in European populations) non-synonymous variants were shared among patients in each family. To prioritize variants for further validation and follow-up, we first searched for variants shared in more than one family; variants in highly conserved regions; those predicted to be damaging by one or more in silico functional models; and variants in genes known to be related to B-cell function and/or disease. We used Ingenuity Variant Analysis to facilitate annotation. We then applied a semi-quantitative scoring algorithm based on these variables to prioritize genes for further evaluation. Although no single gene met all criteria when analyzed across families using this method, we are currently conducting targeted sequencing of 17 highest-scoring genes in an additional 2 WM patients and 92 relatives from these families and 272 other subjects (95 familial WM, 28 nonfamilial WM and 149 informative relatives). Susceptibility gene discovery in this complex disease remains challenging.

Citation Format: Mary L. McMaster, Lynn R. Goldin, Melissa Rotunno, Ji He, Laurie Burdette, Amy Hutchinson, Joseph Boland, Meredith Yeager, Margaret A. Tucker, Stephen J. Chanock, Neil E. Caporaso. Exploration of rare variants from exome sequencing in families with Waldenstrom macroglobulinemia (WM). [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 1300. doi:10.1158/1538-7445.AM2014-1300
Abstract 1302: Genetic analysis in a patient with nine primary malignant neoplasms: A rare case of Li-Fraumeni syndrome
Authors: Li Xiaoyuan , Kang Juan , Pan Qi , Yin Wenwei , Tong Shiwen , Zhao Dachun , Meng Changting , Bai Chunmei , Ren Hong , Ding Keyue
Published: May 2015

The identification of patients with hereditary cancer predisposition syndromes has important clinical implications for early diagnosis, prevention, and treatment. We applied whole exome sequencing to identify mutations underlying a patient diagnosed with Li-Fraumeni syndrome, who had a first-degree family antecedent of breast cancer at age 42 years. Nine primary metachronous malignant neoplasms were developed in this patient within a period of 38 years, including upper lip neuroblastoma (7 years) and rhabdomyosarcoma (7 years), breast cystosarcoma phyllodes carcinoma (28 years), bronchioalveolar cell carcinoma (29 years), breast infiltrating ductal carcinoma (39 years), and thyroid papillary carcinoma (39 years), gastric adenocarcinoma (41 years), maxillary (41 years) and sternal osteosarcoma (43 years), as well as acute myeloid leukemia (45 years). Peripheral blood lymphocyte DNA from the patient was obtained. Exome capture was performed using the SureSelect Human All Exon 51Mb System, and paired-end sequencing was performed using Illumina HiSeq90 platform. A bioinformatics pipeline was used to analyze sequencing reads and call single nucleotide variants (SNVs). We used a candidate strategy to analyze mutations in the case of only a single dominantly affected individual available. Whole-exome sequencing of peripheral blood lymphocyte DNA resulted in an output of 30 GB of raw sequence. After quality control, we obtained clean reads with an average depth of coverage of 105× and 99.4% exome regions were covered at least four times. Analysis of 451,409 SNVs revealed increased genetic risk for some cancers, including genes related to hereditary cancer disposition syndromes [e.g., TP53 (p.P72R and p.R273H), BRCA1, and BRCA2], and genes associated with sporadic cancers. Pathway analysis found that, of 68 genes in p53-signaling pathway, the number of mutated genes (n=14) was significantly higher than expected (P=0.02). Of novel mutations, five nonsense mutations in four genes and 242 missense mutations in 233 genes were discovered. Whole-exome sequencing can identify known and/or novel mutations in cancer susceptibility genes and establish mutation profile in a rare case of Li-Fraumeni syndrome.

Citation Format: Xiaoyuan Li, Juan Kang, Qi Pan, Wenwei Yin, Shiwen Tong, Dachun Zhao, Changting Meng, Chunmei Bai, Hong Ren, Keyue Ding. Genetic analysis in a patient with nine primary malignant neoplasms: A rare case of Li-Fraumeni syndrome. [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 1302. doi:10.1158/1538-7445.AM2014-1302
Abstract 1303: Genetic cancer incidence
Authors: Kim Min Kyu , Bae Soo Youn , Lee Jee Yeon
Published: May 2015

Objective: Hereditary Breast Ovarian Cancer (HBOC) is genetic malignancy associated with BRCA1/2 genes. Selecting confirmative tests for target population are not clearly defined and expensive. We undertook this study to find the detection rate of HBOC among Korean ovarian carcinoma patients based on family history and immunohistochemistry right after by surgeon.

Methods: This study examined 22 patients diagnosed with ovarian carcinomas by single surgeon and counselor. All patients were provided genetic counseling based on immunohistochemistry (IHC) of mismatch repair genes (BRCA1, BRCA2) and 1st, 2nd 3 rd relatives' family history of cancer. Additionally, direct full sequencing to verify germ line mutations was performed to all patients after consent.

Results: Median age was 54.Preop CA 125 was 1824(37.71∼16009). There were 3 refusals after counseling. Patients with family cancer history were 3/21(14%). The number of patients with negative IHC results for BRCA1/BRCA2 was 9 and 6.Both negative patients were 3/21(14%). Both normal staining patients were 4/21(19%).

There were 3/19 patients (16%) with germ line mutations (two BRCA1 and one BRCA2).Variation of unknown significance mutation patients were 5/19(26%).

Conclusion: Our data indicate approximately 17% of patients have a germ line mutation in this study group. Active genetic counseling by surgeon with IHC and family history can help detect HBOC among ovarian carcinoma patients.

Citation Format: Min Kyu Kim, Soo Youn Bae, Jee Yeon Lee. Genetic cancer incidence. [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 1303. doi:10.1158/1538-7445.AM2014-1303
Abstract 1312: Buformin, an anti-diabetic biguanide, inhibits proliferation, invasion and adhesion, and acts synergistically with paclitaxel in endometrial cancer cell lines
Authors: Kilgore Joshua , Jackson Amanda , Qiu Haifeng , Zhou Chunxiao , Gehrig Paola , Bae-Jump Victoria
Published: May 2015

Objectives: Epidemiologic and pre-clinical studies suggest that anti-diabetic biguanide drugs, such as metformin and buformin, may have anti-tumorigenic effects and behave as chemosensitizers. Thus, we evaluated the effects of buformin alone and in combination with paclitaxel on proliferation in human endometrial cancer (EC) cell lines as well as the impact of buformin on apoptosis and adhesion/invasion.

Methods: The ECC-1 and Ishikawa EC cell lines were treated with varying doses of buformin alone and in combination with paclitaxel. Cell growth was determined by MTT assay. Cell cycle progression was assessed by Cellometer. Apoptosis was evaluated by Annexin V-FITC assay. Invasion was demonstrated by transwell invasion assay. Adhesion was assessed by ELISA. Effects on AMPK and S6 expression were documented by Western blotting.

Results: Buformin inhibited proliferation in a dose-dependent manner in the EC cell lines (IC50 1000 μM for ECC-1, 10μM for Ishikawa, p=0.00001-0.0010). Treatment with buformin resulted in G1 arrest in the ECC-1 cell line and G2 arrest in the Ishikawa cell line. Buformin induced apoptosis in the Ishikawa cell line, but not in the ECC-1 cell line. Treatment with buformin resulted in inhibition of adhesion (p=0.42-0.0009) and invasion (p=0.00001-0.227) in both EC cell lines. Western blotting analysis demonstrated that buformin increased phosphorylation of AMPK and decreased phosphorylation of S6. Paclitaxel inhibited proliferation in a dose-dependent manner in both cell lines with IC50 values of 0.1-0.5 nM and 1-5 nM for Ishikawa and ECC-1 cells, respectively. To assess synergy of paclitaxel and buformin, the combination index (CI) was calculated by the method of Chou and Talalay. Simultaneous exposure of cells to various doses of paclitaxel in combination with buformin (0.1-1 μM) resulted in a significant synergistic anti-proliferative effect (CI <1, range 0.009-0.85).

Conclusions: Buformin inhibited proliferation in EC cell lines, via AMPK activation and mTOR pathway inhibition. Inhibition of adhesion/invasion was also seen with buformin. Buformin acted synergistically with paclitaxel to inhibit cell proliferation. More work is needed to determine if buformin and other biguanides alone or in combination with cytotoxic agents will be beneficial in the treatment of women with EC.

Citation Format: Joshua Kilgore, Amanda Jackson, Haifeng Qiu, Chunxiao Zhou, Paola Gehrig, Victoria Bae-Jump. Buformin, an anti-diabetic biguanide, inhibits proliferation, invasion and adhesion, and acts synergistically with paclitaxel in endometrial cancer cell lines. [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 1312. doi:10.1158/1538-7445.AM2014-1312
Abstract 1315: CtIP is regulated by the APC/C-Cdh1 to mediate cell cycle-dependent control of DNA repair
Authors: Boer Harmen R. de , Lafranchi Lorenzo , Neugebauer Christine , Fehrmann Rudolf S.N. , Vries Elisabeth G.E. de , Sartori Alessandro A. , Vugt Marcel A.T.M. van
Published: May 2015

Human cells have evolved elaborate mechanisms for responding to DNA damage to maintain genome stability and prevent carcinogenesis. For instance, the cell cycle can be arrested at different stages to allow time for DNA repair. The APC/C-Cdh1 ubiquitin ligase regulates mitotic exit but is also implicated in the DNA damage-induced G2 arrest. However, it is unknown whether APC/C-Cdh1 also directly participates in DNA repair. Here we show that depletion of Cdh1 in untransformed RPE-1 cells or several cancer cell lines results in increased levels of genomic instability as well as enhanced sensitivity to DNA double-strand break (DSB)-inducing agents. Moreover, Cdh1 depletion results in aberrant homology-directed repair. Using an integrated SILAC proteomics and bioinformatics approach in RPE-1 cells, we identified CtIP as a novel APC/C-Cdh1 target. CtIP interacts with Cdh1 through a conserved KEN box, of which mutation prevents CtIP ubiquitination and impedes CtIP down-regulation both during G1 and after DNA damage in G2. Importantly, the inability of CtIP to interact with Cdh1 causes defective homology-directed repair. Combined, these results show that APC/C-Cdh1 safeguards genome integrity by controlling CtIP stability in a cell cycle- and DNA damage-dependent manner. Supported by European Research Councel grant -2011-293445; Dutch Cancer Society grant RUG-2011-5093; the Vontobel Stiftung and Swiss National Science Foundation grant 31003A_135507.

Citation Format: Harmen R. de Boer, Lorenzo Lafranchi, Christine Neugebauer, Rudolf S.N. Fehrmann, Elisabeth G.E. de Vries, Alessandro A. Sartori, Marcel A.T.M. van Vugt. CtIP is regulated by the APC/C-Cdh1 to mediate cell cycle-dependent control of DNA repair. [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 1315. doi:10.1158/1538-7445.AM2014-1315
Abstract 1319: Vulnerability and resistance in glioblastoma to G2-specific drugs
Authors: Dhruv Harshil D. , Nelson Andrew D. , Armstrong Brock , Boerner Julie L. , Sarkaria Jann N. , Tran Nhan L. , Berens Michael
Published: May 2015

Glioblastoma multiforme (GBM) is the most common and most lethal primary malignant brain tumor, affecting 25,000 patients per year. Approximately 87% of GBMs have abnormalities in the p53 pathway that affects G2 checkpoint. In the absence of p53, Wee1 kinase has been shown to regulate G2 checkpoint upon chemotherapeutic insult. Wee1 inhibits CDC2 function, thereby promoting G2 phase cell cycle arrest, thus preventing damaged cells from entering into premature mitosis without repairing the DNA. We hypothesize that in p53-deficient tumor cells, inhibitors of Wee1 would be effective to heighten DNA damage due to genetic and pharmacologic loss of both the G1 and G2 checkpoints, respectively. MK-1775 (Merck) is a potent and selective small molecule inhibitor of the Wee1 kinase that has demonstrated enhanced cytotoxic effect in combination with several chemotherapeutic agents in preclinical studies in vitro and in vivo. Here, we selected an array of short term cultures of glioma patient derived xenograft (PDX) models (GBM12, GBM43, GBM79, GBM84) and long term established glioma cell lines (U87, SF767, T98G) with aberrant and wild type G1 checkpoint pathways and investigated cytotoxic effects of MK-1775 as a single agent and in combination with Temozolomide (TMZ). Our results demonstrated differential sensitivity to MK-1775 and TMZ as single agents for different glioma cell lines in vitro. However, no correlation between MK-1775, TMZ sensitivity and p53 status of the cell lines was observed. Combination therapy with MK-1775 and TMZ showed a synergistic effect for one PDX model, GBM43, with combination index (CI) of 0.4. Combination therapy of all other glioma cell lines either showed an additive effect or antagonistic effect with CI of >0.75 irrespective of their p53 status. Our results demonstrated that synergistic effects of MK-1775 in combination with chemotherapy (TMZ) are not solely dependent on p53 status of glioma cells and require a more comprehensivce understanding of the molecular mechanism that dictates GBM's dependence on Wee1 for escaping DNA damage therapy. Supported by the Ben & Catherine Ivy Foundation

Citation Format: Harshil D. Dhruv, Andrew D. Nelson, Brock Armstrong, Julie L. Boerner, Jann N. Sarkaria, Nhan L. Tran, Michael Berens. Vulnerability and resistance in glioblastoma to G2-specific drugs. [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 1319. doi:10.1158/1538-7445.AM2014-1319
Abstract 1327: Targeting the RB-pathway in sarcoma: Utility of CDK4/6 inhibitors
Authors: Francis Ashleigh M. , Carey Jason P. , Alexander Angela , Keyomarsi Khandan , Hunt Kelly K.
Published: May 2015

Background: Sarcomas are rare neoplasms of mesenchymal origin. Survival rates have not improved with standard chemotherapy; however, molecular insights have posed new drug targets. Aberrations in the RB-pathway, including loss of RB, CDK4 and cyclin D1 overexpression and p16 loss, have been identified in sarcomas. PD 03322991 (PD) is a second generation CDK4/6 inhibitor with the potential to reactivate the RB-pathway and suppress cell cycle progression. We evaluated the efficacy of PD in a panel of sarcoma cell lines.

Methods: Sarcoma cell lines (HT-1080, SK-LMS-1 and SaOS-2) were evaluated for RB-pathway alterations using western blot. Cells were treated with PD for 1, 2, 4, 6 and 8 days to determine IC50 values by MTT assay. Following the same treatment protocol at IC50 concentrations of PD, cells were harvested for cell cycle analysis, western blot and annexin cell death assay.

Results: HT-1080 and SK-LMS-1 were RB-positive with high expression of phospho-RB; SaOS-2 was RB-negative. CDK4/6 were uniformly expressed. HT-1080 and SK-LMS-1 had p16 loss while SaOS-2 overexpressed cyclin D1. PD was effective in all cell lines. Increasing treatment duration led to reduction of IC50 values. PD caused marked G1 arrest as early as day 1 in RB-positive HT-1080 (86.2% vs 74.2% [vehicle]) and SK-LMS-1 (91.4% vs 67.8% [vehicle]) but not RB-negative SaOS-2 (52.9-59.4% vs 57.0% [vehicle]). Annexin assay demonstrated minimal apoptosis with PD (HT-1080: 1.4-3.0% vs 41.2% [control], SK-LMS-1: 0.6-12.0% vs 23.4% [control], SaOS-2: 3.6-7.3% vs 78.7% [control]).

Conclusions: PD appears to have a cytostatic rather than cytotoxic effect in sarcoma. RB-negativity does not preclude PD efficacy but may require higher IC50 values. In RB-negative sarcomas, PD does not cause cell cycle arrest suggesting an alternate mechanism from reactivation of the RB-pathway is present. PD may serve as a potential therapy for sarcoma and should be studied in combination with other targeted therapies that can take advantage of its ability to cause G1 arrest.

Cell LinePD 991 IC50 Concentration (uM)Day 1Day 2Day 4Day 6Day 8HT-1080∼∼4.31.51.1SK-LMS-1∼∼∼2.41.2SaOS-2∼15.510.17.06.0

Citation Format: Ashleigh M. Francis, Jason P. Carey, Angela Alexander, Khandan Keyomarsi, Kelly K. Hunt. Targeting the RB-pathway in sarcoma: Utility of CDK4/6 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 1327. doi:10.1158/1538-7445.AM2014-1327
Abstract 1330: Immunotherapy targeting folate receptor induces autophagy in ovarian cancer
Authors: Wen Yun-Fei , Graybill Whitney Spannuth ,   Department of Gyneocologic Oncology, Sood Anil
Published: May 2015

Objective Cancer cells are highly dependent on folate metabolism for replication of DNA, making these cells susceptible to drugs that inhibit folate metabolism. Herein, we investigated the functional mechanism underlying blockade of folate receptor (FR) in human ovarian cancer with MORAb-003 antibody, a humanized monoclonal antibody directly against folate receptor 1 (FRα).

Methods We first used FACS analysis and real-time PCR to compare the expression of FRα in several human ovarian cancer cells. We then examined the tumor growth in the orthotopic models of human ovarian cancer cells SKOV3 and A2780. Consequently, we performed in vitro genomic and proteomic profiling in these cells to identify the functional mechanism of MORAb-003.

Results Among 7 human ovarian cancer cell lines, SKOV3ip1 cells have the highest levels of FRα and A2780 cells have the lowest. In vivo, MORAb-003 monotherapy led to significantly decreased tumor growth in the SKOV3ip1 model, but not in the A2780 model. Combination of MORAb-003 and docetaxel resulted in significantly greater reduction in tumor growth in the SKOV3ip1 model, but not in the A2780 model. MORAb-003 reduced proliferation but had no significant effect on apoptosis. Reverse-phase array analysis in SKOV3 cells revealed MORAb-003 up-regulated autophagic factors including PEA-15, Beclin-1, and ATG3 and down-regulated mTOR. cDNA microarray further showed that MORAb-003 up-regulated the expression of BECN1, ATG8, ATG3 and LC3B. Levels of Beclin-1 and LC3B were substantially increased in SKOV3 tumors treated with MORAb-003 alone or in combination with docetaxel, but not in A2780 tumors. Using FACS analysis with acridine orange (AO) staining, we observed significantly more AO positive population in SKOV3 cells treated with MORAb-003 alone (16.9%) or MORAb-003+docetaxel (21.2%) than in cells treated with control IgG (5.24%). Such differences were not observed in A2780 cells. Finally, we measured conversion of LC3 from isoform I to isoform II in cells treated with MORAb-003, and observed the significantly increased LC3II in SKOV3 cells but not in A2780 cells.

Conclusion Taken together, our results indicate that MORAb-003 results in prominent anti-tumor activity through inducing sustained autophagy in ovarian cancer cells.

Citation Format: Yun-Fei Wen, Whitney Spannuth Graybill, Department of Gyneocologic Oncology, Anil Sood. Immunotherapy targeting folate receptor induces autophagy in 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 1330. doi:10.1158/1538-7445.AM2014-1330
Abstract 1332: Inhibition of G9a induces DUSP4-dependent autophagic cell death in head and neck squamous cell carcinoma
Authors: LI KAI-CHUN , HUA KUO-TAI , TAN CHING-TING , KUO MIN-LIANG
Published: May 2015

Emerging evidence indicates that alteration of epigenetic regulation might be a key event for cancer progression. Abnormal expression of histone methyltransferase G9a, which contributes to transcriptional repression of tumor suppressor genes, has been implicated in maintaining cancer cells proliferation. Recent study suggests that inhibition of G9a reduces cancer cell growth. However, its molecular process has not been fully explored. In this study, we show that G9a expression is positively correlated to poor prognosis in head and neck squamous cell carcinoma (HNSCC) patients. Knockdown of G9a or inhibition of its catalytic activity by bix-01294 in HNSCC cells reduced proliferation, colony formation and anchorage-independent growth without inducing necrosis or apoptosis. Instead, complementary approaches including immunoblot analysis, LC3-GFP-fluorescent microscopy and morphologic study by electron microscopy revealed inhibition of G9a inducing autophagy. Pre-treatment with autophagy inhibitor 3-methyladenine (3-MA) or chloroquine (CQ) antagonized the inhibition effect of bix-01294 on cells viability, demonstrating that autophagic cell death is the major consequences in G9a suppressed cells. Mechanistic investigation suggested that ERK phosphatase, DUSP4, may be a downstream target of G9a. Knockdown of G9a and then suppression of DUSP4 expression restored ERK phosphorylation and colony formation but inhibits autophagy induced by G9a silencing. This effect was further reversed by treatment with MAPK/ERK selective inhibitor U0126. Moreover, knockdown of G9a resulted in growth inhibition and autophagy in orthotopic tumor model in mice. Taken together, our results not only point out the mechanism of autophagic cell death induced by inhibition of G9a but also pave a way for new targeting therapy utility for HNSCC in clinical.

Citation Format: KAI-CHUN LI, KUO-TAI HUA, CHING-TING TAN, MIN-LIANG KUO. Inhibition of G9a induces DUSP4-dependent autophagic cell death 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 1332. doi:10.1158/1538-7445.AM2014-1332
Abstract 1333: Combining Afatinib and Cetuximab synergistically increases their cytotoxicity for EGFR T790M-harboring cells
Authors: Tanaka Nobuyuki , Watanuki Zenta , Fukuhara Tatsuro , Maemondo Makoto
Published: May 2015

Epidermal growth factor receptor (EGFR) is an important therapeutic target for non-small cell lung cancers (NSCLCs). Tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib, are effective for patients with EGFR-activating mutations. However, these patients eventually develop resistance, usually through a secondary EGFR mutation, T790M: substitution of methionine for threonine at position 790 in the kinase domain. While second-generation irreversible TKIs have higher affinities for the double-mutant EGFRs, their therapeutic effect leaves room for improvement. Combining a second-generation TKI like afatinib with an anti-EGFR monoclonal antibody has been shown to improve clinical outcomes, although the mechanism is not fully understood. To investigate this mechanism, we used human erythroleukemia K562 cells, which are EGFR-negative, to establish cells with known EGFR mutations. Two activating EGFR mutations, Ex19del and L858R, were sensitive to first-generation TKIs. Acquisition of resistance to these TKIs was confirmed by adding the second EGFR mutation, T790M. Double-mutant EGFRs were moderately sensitive to afatinib, but were only minimally affected by the monoclonal anti-EGFR antibody cetuximab. Combining afatinib and cetuximab synergistically increased cytotoxity for K562 cells carrying double-mutant EGFRs. Apoptosis in these cells was preceded by induction of the pro-apoptotic protein BIM (Bcl2L11) and activation of Caspase-3 and PARP. Afatinib induced autophagy in these cells correlated with AKT and ERK1/2 signaling in the course of treatment. Accordingly, we found that cetuximab enhanced the afatinib-induced cytotoxicity through the autophagic cell death pathway. Our results indicate that two distinct mechanisms contribute to the synergistic effect exerted by afatinib and cetuximab against NSCLCs.

Citation Format: Nobuyuki Tanaka, Zenta Watanuki, Tatsuro Fukuhara, Makoto Maemondo. Combining Afatinib and Cetuximab synergistically increases their cytotoxicity for EGFR T790M-harboring 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 1333. doi:10.1158/1538-7445.AM2014-1333
Abstract 1335: Lack of casein kinase 1 delta induces DNA damage, inhibition of mTORC1 signaling and nucleophagy
Authors: Greer Yoshimi Endo , Gao Bo , Yang Yingzi , Rubin Jeffrey S.
Published: May 2015

Casein kinase 1 delta (CK1δ) is a conserved serine/threonine protein kinase that regulates diverse cellular processes including vesicle trafficking, cell cycle progression, chromosomal segregation, DNA repair, circadian rhythm, neurite outgrowth and ciliogenesis. CK1δ has been reported to phosphorylate Mdm2, the E3 ubiquitin ligase for p53, resulting in Mdm2 ubiquitination/degradation and concomitant accumulation of p53, the latter functioning as a mediator of cell cycle checkpoints and DNA repair. Mice that lack Csnk1d exhibit a perinatal lethal phenotype and typically weigh 30-50% less than their wild type littermates. The exact causes of death and small size are unknown. We hypothesized that the absence of CK1δ initiates cellular stresses that adversely affect cell survival. To examine this idea, we collected mouse embryonic fibroblasts (MEFs) from mice homozygous for a Csnk1d floxed allele, and generated MEF Csnk1d null cells by infection with adenovirus expressing Cre. Endogenous p53 protein level was significantly lower in MEF Csnk1d null cells relative to MEF Ctl cells infected with adenovirus-GFP. Substantial DNA damage also was detected in MEF Csnk1d null cells, as measured by γ-H2AX staining. Flow cytometric analysis revealed that early passage (P3) MEF Csnk1d null cells exhibited a larger sub-G0 fraction (64%) than MEF Ctl cells (34%). The sub-G0 fraction decreased sharply with increasing passage number, becoming negligible by P10. In addition to the decline in sub-G0 fraction, we observed a transient population of MEF Csnk1d null cells with multiple DAPI-stained micronuclei containing both γ-H2AX and membranes positive for the autophagosomal marker LC3 and lysosomal marker LAMP1. These features were preceded by and coincided with mTORC1 pathway inactivation, as indicated by low concentrations of phospho-Akt, phospho-mTOR and phospho-S6K, consistent with the view that mTORC1 inhibition facilitates the induction of autophagy. Subsequently, these differences between MEF Csnk1d null and MEF Ctl cells subsided. Our findings demonstrate that loss of CK1δ expression in proliferating MEF cells results in DNA damage and initially a large sub-G0 peak, followed by a wave of nucleophagy and ultimately cell survival in culture. Ongoing investigation will explore whether similar processes occur in embryonic tissues from Csnk1d null mice and contribute to their pathologic phenotype.

Citation Format: Yoshimi Endo Greer, Bo Gao, Yingzi Yang, Jeffrey S. Rubin. Lack of casein kinase 1 delta induces DNA damage, inhibition of mTORC1 signaling and nucleophagy. [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 1335. doi:10.1158/1538-7445.AM2014-1335
Abstract 1338: Blockage of nucleocytoplasmic transport by wheat germ agglutinin (WGA) induces autophagy and cell death
Authors: Tsai Tsung-Lin , Hung Chun-Hua , Wang Hao-Chen , Shieh Dar-Bin , Su Wu-Chou , Lin Chien-Chung
Published: May 2015

The nuclear pore complex (NPC) is a primary conduit for cellular communication between the nucleus and cytoplasm, which is essential for cellular survival and proliferation. During carcinogenesis, many oncoproteins are activated and have to send their signals into nucleus to regulate gene expression. In response to the signals, many genes are upregulated, which results in transcription of many RNA. Because both inward of transcriptional factors and outward of RNAs require interaction of these molecules with NPC, blockage of NPC may provoke serious cellular distress. Wheat germ agglutinin (WGA), a lectin that specifically binds to N-acetyl-d-glucosamine (GlcNAc) and N-acetyl-d-neuraminic acid (NeuNAc) residues on the cell surface, was reported to disrupt the function of NPC via interaction with POM121. We found that FITC-WGA had located around the cell membrane 0.5 hour after administration to culture media and reached the paranuclear region 4 hours later. The accumulation of WGA around the nucleus subsequently induced a nucleocytoplasmic transport blockage that prevented export of tRNA and import of signaling proteins (NF-κB and Nrf2) into the nucleus. Beyond the concentration of 5 μg/ml, WGA dose- and time-dependently induced cell death in HeLa, SiHa and CaSKi cell lines. The SiHa cells had more intake of WGA than HeLa and CaSKi cells did and became more sensitive to WGA treatment compared to the other two cells. After treatment with WGA in the three cells, we observed that the expression of autophagic markers (ATG5, ATG7 and LC3) are induced in 24 hours and last to 96 hours. On the contrary, the apoptotic protein caspases 3 and downstream substrates, such as poly(ADP-ribose) polymerase (PARP) were only marginally induced. Furthermore, Transmission Electron Microscopy (TEM) imaging disclosed multi-membrane vesicles (autophagosomes and autolysosomes) inside the cytoplasm after WGA treatment, confirming that the autophagy response was fully induced. These findings suggest that blockage of NPC by WGA may induced autophagy-related cell death and targeting NPC may be warranting further development into a new anti-cancer modality.

Citation Format: Tsung-Lin Tsai, Chun-Hua Hung, Hao-Chen Wang, Dar-Bin Shieh, Wu-Chou Su, Chien-Chung Lin. Blockage of nucleocytoplasmic transport by wheat germ agglutinin (WGA) induces autophagy and cell death. [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 1338. doi:10.1158/1538-7445.AM2014-1338
Abstract 1339: Endoplasmic reticulum stress mediates Tanshinone I induced apoptosis in mesothelioma cells
Authors: Lee JiHyun , Kim Bonglee
Published: May 2015

Malignant Mesothelioma (MM) is a highly aggressive tumor caused by asbestos exposure with poor survival rate. Here, we investigated anti-cancer mechanism of Tanshinone I on mesothelioma cells. Tanshinone I exerted significant cytotoxicity in a time and dose dependent manner in mesothelioma cell lines such as MSTO-211H, H28, and H2452 cells, but not in MeT-5A cells. Also, Tanshinone I increased sub-G1 population and TUNEL positive stained cells in three mesothelioma cells. Consistently, Tanshinone I cleaved poly(ADP)-ribose polymerase (PARP) and attenuated the expression of pro-caspase9, pro-caspase3 and Bcl2 as well as the phosphorylation of JNK and PI3K in three mesothelioma cells. Furthermore, Tanshinone I exerted significant cytotoxicity in a time and dose dependent manner in mesothelioma up-regulated the expression of endoplasmic reticulum (ER) stress markers such as CHOP, GRP78, ATF4, and IRE-1 in three mesothelioma cells. The apoptotic ability of Tanshinone I to cleave PARP and activate caspase 9/3 in in three mesothelioma cells. Overall, these findings suggest that Tanshinone I exhibits anti-tumor effect in MSTO-211H, H28, and H2452 cells via up-regulation of ER stress signaling as a potent anticancer agent for the mesothelioma cells.

Citation Format: JiHyun Lee, Bonglee Kim. Endoplasmic reticulum stress mediates Tanshinone I induced apoptosis in mesothelioma 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 1339. doi:10.1158/1538-7445.AM2014-1339