bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 577: DNA replication origin of human alphoid centromere regions .
Authors: Erliandri Indri , Fu Haiqing , Kim Jung-Hyun , Aladjem Mirit , Earnshaw William , Nagaraja Ramaiah , Schlessinger David , Larionov Vladimir
Published: August 2014

The human centromere DNA consist of 171 bp AT-rich-alpha satellite DNA that tandemly arranged into higher order repeat units that is diverged at each centromere, extend from 200kb to 4MB. As the chromosomal site that regulates accurate chromosome segregation, maintaining the correct and coordinated DNA replication prior to cell division is critical. This process should ensure that whole genetic information transferred to daughter cell faithfully. Irregularities on replication process are leading to the mitosis defect and apoptosis. Flanking regions of alpha-satellite DNA arrays are organized in heterochromatin domains that are co-localized with other types of centromeric repeats, i.e. satellite II and gamma-satellite DNA. Previous studies indicate that centromeric DNA replicates in the middle to late S-phase. However little is known what determines activities and positions of replicators on chromatin domains. In this work, we addressed the mechanism of replication on the centromere and other repeat sequences on pericentromeric regions using two different approaches: i) origin mapping using nascent-strand DNA and ii) DNA-protein interaction using chromatin-immunoprecipitation (ChIP) assay. Our results detect active origin of replication within alpha-satellite DNA monomers of centromere, as well as on gamma-satellite repeats. In contrast, satellite II repeats representing centromere-adjacent heterochromatin do not exhibit a significant activity as origin of replication. The accumulation of modified-Histone proteins indicated dynamic activity of DNA arrays. Moreover, DNA replication proteins are formed complex with centromere proteins, presented direct evident on DNA replication activity on centromere. In summary, data indicated position of replicator within alphoid centromere and other large tandem repeat arrays on pericentromeric region with condense chromatin structure, thus suggested role of epigenetic modifications on DNA arrays propagation.

Citation Format: Indri Erliandri, Haiqing Fu, Jung-Hyun Kim, Mirit Aladjem, William Earnshaw, Ramaiah Nagaraja, David Schlessinger, Vladimir Larionov. DNA replication origin of human alphoid centromere regions . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 577. doi:10.1158/1538-7445.AM2013-577
Abstract 595: A cell-permeant amiloride derivative induces caspase-independent, AIF-mediated programmed necrotic death of breast cancer cells.
Authors: Leon Leonardo J. , Pasupuleti Nagarekha , Gorin Fredric , Carraway Kermit
Published: August 2014

Amiloride is a potassium-sparing diuretic that has been used as an anti-kaliuretic for the chronic management of hypertension and heart failure. Several studies have identified a potential anti-cancer role for amiloride, however the mechanisms underlying its anti-tumor effects remain to be fully delineated. Our group previously demonstrated that amiloride triggers caspase-independent cytotoxic cell death in human glioblastoma cell lines but not in primary astrocytes. To delineate the cellular mechanisms underlying amiloride's anti-cancer cytotoxicity, cell permeant and cell impermeant derivatives of amiloride were synthesized that exhibit markedly different potencies in cancer cell death assays. Here we compare the cytotoxicities of 5-benzylglycinyl amiloride (UCD38B), and its free acid 5-glycinyl amiloride (UCD74A) toward human breast cancer cells. UCD74A exhibits poor cell permeability and has very little cytotoxic activity, while UCD38B is cell permeant and induces the caspase-independent death of proliferating and non-proliferating breast cancer cells. UCD38B treatment of human breast cancer cells induces autophagy, reflected in LC3 conversion, however this does not appear to be the cause of cell death. Electron microscopy reveals that UCD38B induces a dramatic swelling of intracellular organelles such as the endoplasmic reticulum and mitochondria. Surprisingly UCD38B, but not UCD74A, induces efficient AIF translocation from the mitochondria to the nucleus, and AIF function is necessary for the efficient induction of cancer cell death. Our observations indicate that UCD38B induces programmed necrosis through AIF translocation, and suggest that cytosolic accessibility may facilitate drug action.

Citation Format: Leonardo J. Leon, Nagarekha Pasupuleti, Fredric Gorin, Kermit Carraway. A cell-permeant amiloride derivative induces caspase-independent, AIF-mediated programmed necrotic death of breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 595. doi:10.1158/1538-7445.AM2013-595

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 601: Antioxidant inhibition of GSK-3β stabilizes anti-apoptotic factor Mcl-1 leading to decreased apoptosis and increased cell survival.
Authors: Niture Suryakant K. , Jaiswal Anil K.
Published: August 2014

Antioxidant-mediated stabilization of Nrf2 is known to up-regulate anti-apoptotic factors Bcl-2 and Bcl-xL that leads to reduced apoptosis and increased cell survival. In this report we investigated and found that anti-apoptotic factor Mcl-1 is coordinately induced with Bcl-2 and Bcl-xL factors in response to antioxidants. However, unlike Bcl-2 and Bcl-xL, Nrf2 did not control antioxidant-mediated up-regulation of Mcl-1. Further studies revealed that ERK1/2 phosphorylation of Mcl-1T163 primes Mcl-1 for phosphorylation by GSK3β at position S159. This modification degrades Mcl-1 and contributes to the maintenance of physiological level of Mcl-1 and cellular apoptosis. Exposure of Hep-G2 cells to antioxidant tert-butyl hydroquinone (t-BHQ) for 4-16h led to time dependent Akt-mediated inactivation of GSK3β, decreased Mcl-1S159 phosphorylation resulting in stabilization of Mcl-1 protein. Antioxidant-mediated increased Mcl-1 heterodimerized with Bim and Bax and down regulated cellular apoptosis. The contribution of antioxidant increased Mcl-1 in reduced apoptosis was further confirmed by siRNA-mediated inhibition of Mcl-1 that increased susceptibility to etoposide-mediated cell death and reduced cell survival through the activation of caspases 3/7. These results provided evidence that antioxidant control of GSK-3β led to increase in Mcl-1 that contributed to decreased apoptosis and increased cell survival. The results collectively suggest that antioxidant stabilized Nrf2 and GSK3β both through distinct mechanisms lead to increased anti-apoptotic factors Bcl-2, Bcl-xL and Mcl-1 that reduces apoptosis and promotes cell survival.

Citation Format: Suryakant K. Niture, Anil K. Jaiswal. Antioxidant inhibition of GSK-3β stabilizes anti-apoptotic factor Mcl-1 leading to decreased apoptosis and increased cell survival. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 601. doi:10.1158/1538-7445.AM2013-601
Abstract 603: CAL-101 acts synergistic with arsenic trioxide in chronic lymphocytic leukemia by preventing p21 phosphorylation through AKT1.
Authors: Merkel Olaf , Sifft Eveline , Schweiggl Simone , Asslaber Daniela , Wacht Nathalie , Greil Richard
Published: August 2014

Chronic lymphocytic leukemia (CLL) is the most abundant leukemia of the elderly in the Western world and characterized by the accumulation of CD5/CD19/CD23 positive cells in the peripheral blood, lymph nodes and spleen. It has a very heterogeneous course of disease with patients living longer than 10 years after diagnosis and others needing immediate treatment. CLL cells of the latter often bear aberrations in the p53 tumor suppressor gene. We have shown before that arsenic trioxide preferentially induces apoptosis in CLL cells deficient for p53 (n = 51, P = 0.029). We now reproduced this finding in mouse embryo fibroblasts (MEF) that are p53 deficient. Next we wanted to clarify the role of p53 downstream targets p21 and PUMA and found that p21 but not PUMA knockout led to increased arsenic trioxide susceptibility. When localized in the cytosol p21 has been described to have and anti-apoptotic function and accordingly is associated with negative prognosis in breast, ovarian and prostate cancer. To clarify p21’s localization in CLL cells we used differential detergent cell fractionation and demonstrate that upon arsenic trioxide treatment p21 is induced, but accumulates in the cytoplasm. This is in contrast to fludarabine treated cells, where p21 is strongly induced and shuttles to the nucleus. Moreover, we show here that arsenic trioxide leads to transient AKT activation and AKT phosphorylation of p21 has been shown to promote cytosolic localization. We next hypothesized that by combining the PI3Kdelta/AKT inhibitor CAL-101 with arsenic trioxide we might prevent cytosolic accumulation of p21. Recently, the idea of therapeutic inhibition of tyrosinse kinases in the B-cell receptor pathway has gained new momentum through pivotal phase-2 clincal studies with CAL-101 and ibrutinib targeting the PI3Kdelta- and the Bruton's tyrosine-kinase, respectively. We show here, that the PI3K inhibitor CAL-101 abrogates AKT activation through arsenic trioxide. This leads to strongly, synergistic apoptosis induction when CAL-101 and arsenic trioxide are combined irrespective of p53 status of CLL cells. Thus, we think that these findings not only shed light on the role of p21 in apoptosis induction in CLL, but open the path to novel therapeutic combination regimens.

Citation Format: Olaf Merkel, Eveline Sifft, Simone Schweiggl, Daniela Asslaber, Nathalie Wacht, Richard Greil. CAL-101 acts synergistic with arsenic trioxide in chronic lymphocytic leukemia by preventing p21 phosphorylation through AKT1. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 603. doi:10.1158/1538-7445.AM2013-603
Abstract 61: Use of two-color fluorometric real-time PCR to develop molecular diagnostic tests with intrinsic quality control that augment cytomorphologic diagnosis of lung cancer.
Authors: Yeo Jiyoun , Crawford Erin L. , Blomquist Thomas M. , Stanoszek Lauren M. , Dannemiller Rachel E. , Jordan Laura , Zyrek Jill , de la Casas Luis , Willey James C.
Published: August 2014

Quantitative RT-PCR tests that measure transcript abundance of selected genes in clinical specimens promise to improve cancer diagnostic accuracy and enable “personalized medicine” through selection of the most effective treatment for each cancer. However, most clinical samples are Formalin-Fixed, Paraffin-Embedded (FFPE) and these yield sub-optimal RNA integrity. Thus, there is a need for qPCR tests with increased robustness that also have intrinsic quality control and low cost. To address this need, we developed qPCR methods that enable simultaneous measurement of each target gene and reference gene transcript relative to a known number of gene-specific internal standard (IS) molecules using two-color fluorometric analysis on real-time platform. For each gene, a competitive template IS was synthesized containing 4-6 nucleotide changes relative to the native template (NT). During PCR, NT or IS signal was quantified with sequence-specific FAM-labeled probe or Quasar 670-labeled probe, respectively. To optimize measurement sensitivity for analysis of highly degraded FFPE samples; a) each RNA sample was reverse transcribed (RT) with gene-specific primer and b) cDNA was pre-amplified with 18 PCR cycles in the presence of internal standards. Results obtained with pre-amp or no pre-amp conditions were highly correspondent.

We conducted a validation study of this assay in 38 samples (10 malignant and 10 benign surgical FFPE tissues and 13 malignant and 5 benign fine needle aspirate (FNA) samples. Consistent with previous results in fresh frozen surgical samples, the lung cancer diagnostic test (LCDT) optimal cut-off value discriminated malignant from non-malignant tissues (p = 0.0009), had 92.9% specificity and 75.0% sensitivity, and a receiver operator characteristic area under the curve of 0.87 (95% confidence interval 0.74-0.99). Based on these data, we expect that this quality-controlled two color fluorometric qPCR approach will enable reliable analysis of the LCDT and other promising qPCR-based molecular diagnostic tests in small degraded RNA extracted from clinical FFPE and FNA cell block FFPE samples.

Citation Format: Jiyoun Yeo, Erin L. Crawford, Thomas M. Blomquist, Lauren M. Stanoszek, Rachel E. Dannemiller, Laura Jordan, Jill Zyrek, Luis de la Casas, James C. Willey. Use of two-color fluorometric real-time PCR to develop molecular diagnostic tests with intrinsic quality control that augment cytomorphologic diagnosis of lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 61. doi:10.1158/1538-7445.AM2013-61
Abstract 615: Knock-down of base excision repair genes increases susceptibility to organophospate pesticide toxicity.
Authors: Saulsbury Marilyn D. , Heyliger Simone , Li Feng , Parker-Johnson KiTani A.
Published: August 2014

Organophosphates are chemicals that inhibit cholinesterases and are employed widely as pesticides. Concerns are increasing regarding the relative safety of these chemicals to the environment. Recent studies suggest organophosphate exposure is associated with increased expression of fragile sites and DNA damage at concentrations that are not associated with cholinesterase inhibition. Chronic exposure to the organophosphate has been associated with high incidences of prostate cancer in farm workers as well as leukemia and non-Hodgkin lymphoma in adults and children. Moreover, a higher degree of DNA damage has been reported in pesticide applicators that have polymorphisms in the base excision repair genes XRCC1 and OGG1. Our laboratory reports that cells deficient in BER enzymes APE1 and OGG1 exhibit a higher cytotoxicity when exposed, in a dose-dependent manner, to organophosphates Chlorpyrifos, Chlorpyrifos-oxon and Isofenphos. However, it should be noted that other organophosphates such as Diazinon and Dichlorvos had very little effects on cellular viability within the APE1 and OGG1 deficient cells. It should be noted that Chlorpyrifos, Chlorpyrifos-oxon and Isofenphos also produce significant oxidative stress in cells. Our data is consistent with previous studies which report a higher degree of DNA damage has been reported in pesticide applicators that have polymorphisms in the base excision repair genes XRCC1 and OGG1. Moreover, our data further suggests that only certain organophosphates, particularly those that induce oxidative stress, may be responsible for toxicity in APE1 and OGG1 deficient cells.

Citation Format: Marilyn D. Saulsbury, Simone Heyliger, Feng Li, KiTani A. Parker-Johnson. Knock-down of base excision repair genes increases susceptibility to organophospate pesticide toxicity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 615. doi:10.1158/1538-7445.AM2013-615
Abstract 617: High-throughput screens identify PARP inhibition as being synthetically lethal with ERCC1 deficiency in non-small cell lung cancer cell lines.
Authors: Postel-Vinay Sophie C. , Bajrami Ilirjana , Friboulet Luc , Aarts Marieke , Fontebasso Yari , Andre Fabrice , Olaussen Ken , Soria Jean-Charles , Lord Chris , Ashworth Alan
Published: August 2014

Background: Non-small cell lung cancer (NSCLC) harbors frequent DNA-repair defects that represent therapeutic opportunities. Notably, Excision Repair Cross-Complementation group 1 (ERCC1) deficiency has been described in a subset of tumors and correlated with platinum sensitivity. The use of mechanism-based approaches, such as synthetic lethality, could allow targeting more selectively such DNA repair defects, with reduced systemic toxicity.

Methods: To identify ERCC1-selective synthetic lethal interactions, we adopted an integrated functional and molecular profiling approach. An isogenic model of ERCC1-deficient NSCLC cells, consisting of one parental ERCC1-proficient cell line and 3 ERCC1-deficient clones, was functionally profiled by high throughput drug sensitivity screening using a library containing commonly used oncology agents as well as a large number of in-development targeted agents. This was integrated with data from molecular profiling, including high throughput siRNA screens.

Results: The comparison of functional viability profiles for ERCC1-deficient models enabled the identification of synthetic lethal effects that could represent therapeutic opportunities for ERCC1-deficient tumors. Notably, ERCC1-deficient cells showed increased sensitivity to several PARP inhibitors, representing promising preliminary results that could be translated in the clinical setting. Mechanistic studies revealed a significant delay in double-strand break repair associated with a G2/M arrest following PARP inhibitor treatment in the ERCC1-deficient population only. Further in vitro work is providing deeper insight into the mechanisms underlying this PARP inhibitors sensitivity and in vivo work is underway.

Conclusions: ERCC1-deficiency sensitizes human NSCLC cell lines to PARP inhibitors. In vitro and in vivo revalidations of these results and now underway to further dissect the molecular mechanisms responsible for these ERCC1-deficient selective effects, which could represent therapeutic opportunities.

Citation Format: Sophie C. Postel-Vinay, Ilirjana Bajrami, Luc Friboulet, Marieke Aarts, Yari Fontebasso, Fabrice Andre, Ken Olaussen, Jean-Charles Soria, Chris Lord, Alan Ashworth. High-throughput screens identify PARP inhibition as being synthetically lethal with ERCC1 deficiency in non-small cell lung cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 617. doi:10.1158/1538-7445.AM2013-617
Abstract 622: Degradation of APE1 by the E3 ubiquitin ligase Parkin.
Authors: Scott Timothy L. , Rangaswamy Suganya , Dhar Bithika , Zhi LianTeng , Bueler Hansruedi , Izumi Tadahide
Published: August 2014

The mammalian apurinic/apyrimidinic endonuclease 1 (APE1/Ref1) is a DNA repair/redox regulator protein of which cytoplasmic accumulation has been associated with tumorigenesis and poor survival outcome. APE1 is degraded through the ubiquitin/proteasome pathway and previous studies reported APE1 ubiquitination by the E3 ubiquitin ligases MDM2 and UBR3. Here we report that Parkin efficiently polyubiquitinated and degraded APE1. Parkin, a product of the PARK2 gene, is responsible for a type of early onset Parkinson's disease (PD) and has been identified as a tumor suppressor. Expression of Parkin and Pink1 enhanced APE1 ubiquitination and markedly decreased endogenous APE1 in A549, a lung adenocarcinoma cell line. APE1 degradation was blocked by incubation of the cells with MG132, a potent 26S proteasome inhibitor, and by a PD associated point mutation in the Parkin RING domain. The activity of PTEN induced-kinase 1 (Pink1) was required for APE1 degradation. Co-immunoprecipitation revealed that APE1 interacted with Parkin in the presence of Pink1. These results suggest that APE1 stability is dynamically regulated by Parkin in the cytoplasm through the N-terminal APE1 domain.

Citation Format: Timothy L. Scott, Suganya Rangaswamy, Bithika Dhar, LianTeng Zhi, Hansruedi Bueler, Tadahide Izumi. Degradation of APE1 by the E3 ubiquitin ligase Parkin. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 622. doi:10.1158/1538-7445.AM2013-622
Abstract 624: Novel interaction of RECQ1 with Ku70/80 and its potential role in non-homologous end joining.
Authors: Parvathaneni Swetha , Brosh Robert M. , Sharma Sudha
Published: August 2014

Genomic instability is a known precursor to cancer and aging. The RecQ helicases are a highly conserved family of DNA-unwinding enzymes that play key roles in maintaining genome stability in all living organisms. Human RecQ homologs include RECQ1, BLM, WRN, RECQL4, and RECQ5β, three of which have been linked to disease with elevated risk of cancer and growth defects (Bloom Syndrome and Rothmund-Thomson Syndrome) or premature aging (Werner Syndrome). RECQ1, the first RecQ helicase discovered and the most abundant in human cells, is the least well understood of the five human RecQ homologs. We have previously described that knockout of RECQ1 in mice or knockdown of its expression in HeLa cells results in elevated frequency of spontaneous sister chromatid exchanges, chromosomal instability, increased load of DNA damage and heightened sensitivity to ionizing radiation. We have now obtained evidence implicating RECQ1 in the nonhomologous end-joining pathway of DNA double strand break repair. We show that RECQ1 interacts directly with the Ku70/80 subunit of the DNA-PK complex, and depletion of RECQ1 results in reduced end-joining in cell free extracts. In vitro, RECQ1 binds and unwinds the Ku70/80 bound partial duplex DNA substrate efficiently. Linear DNA is co-bound by RECQ1 and Ku70/80, and DNA binding by Ku70/80 is modulated by RECQ1. Collectively, these results provide the first evidence for a role of RECQ1 in nonhomologous end-joining, perhaps through interaction with Ku70/80.

Citation Format: Swetha Parvathaneni, Robert M. Brosh, Sudha Sharma. Novel interaction of RECQ1 with Ku70/80 and its potential role in non-homologous end joining. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 624. doi:10.1158/1538-7445.AM2013-624
Abstract 629: Oddities of DNA damage signaling in mitotic human cells revealed by super-resolution microscopy.
Authors: Mistrik Martin , Bartek Jiri
Published: August 2014

Using an optimized cell-spreading technique combined with immunofluorescence (IF) and super-resolution microscopy we examined in greater detail DNA lesions induced within human mitotic chromosomes. We found some major differences in DNA damage signaling of mitosis-associated lesions induced by different genotoxic insults, such as inducers of DNA replication stress and ionizing radiation. While the former source of DNA damage represents a prominent inducer of genomic instability - a hallmark of cancer, the latter treatment is commonly used in cancer therapy. Differences in the DNA damage signal spreading and participation of various DNA damage-responsive proteins differentially recruited to sites of the DNA lesions are presented and discussed, including our innovative techniques developed for quantitative IF analysis of mitotic cells.

Citation Format: Martin Mistrik, Jiri Bartek. Oddities of DNA damage signaling in mitotic human cells revealed by super-resolution microscopy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 629. doi:10.1158/1538-7445.AM2013-629
Abstract 656: Promoter hypermethylation and silencing of tumor suppressor genes RASSF1A, WIF1, SLIT2 and ROBO1 in triple negative breast carcinomas.
Authors: Alvarez Carolina , Garrido Gloria , Gajardo Patricia , Quiroz Alonso , Cornejo Valeria , Fernandez Wanda , Camus Mauricio , Carvallo Pilar
Published: August 2014

Triple negative breast cancers represent 10-17% of all breast carcinomas. They display a high histological grade, a more aggressive clinical behavior and they lack any specific treatment, thus are associated to a bad prognosis. As other cancer types, this cancer is caused by sequential accumulation of mutations and genomic alterations involving tumor suppressor genes and oncogenes. In this matter, inactivation of tumor suppressor genes may occur by deletion, promoter hypermethylation or point mutations. In previous study by array CGH we found several tumor suppressor candidate genes deleted in a hereditary breast cancer set of tumors. In addition we studied 4 of these tumor suppressor genes RASSF1A, WIF1, SLIT2 and ROBO1 in a non triple negative set of breast cancer tumors, and found a high frequency of promoter methylation, statistically associated with loss of protein expression for RASSF1A and WIF1. In the present study we aimed to extend the analysis to triple negative breast cancer tumors due to their clinical relevance. Methylation analysis through MS-PCR revealed that RASSF1A promoter was hypermethylated in 52.9% of triple negative tumors, and presents loss of expression in 80.6% of the tumors. WIF1 promoter was hypermethylated in 89.1% and its protein expression was downregulated in 63.1% of the tumors. For SLIT2 we found frequent promoter hypermethylation among tumors (82.6%) showing loss of expression in 88% of the tumors. MS-PCR analyses were not informative for ROBO1, but protein expression was reduced in 49% of all tumors. Our results show that a high percentage of tumors present silencing of RASSF1A, WIF1, SLIT2 and/or ROBO1, and therefore implying the impairment of the pathways they represent. This findings support these genes may have an important role in triple negative breast tumor development. Fondecyt 1080595.

Citation Format: Carolina Alvarez, Gloria Garrido, Patricia Gajardo, Alonso Quiroz, Valeria Cornejo, Wanda Fernandez, Mauricio Camus, Pilar Carvallo. Promoter hypermethylation and silencing of tumor suppressor genes RASSF1A, WIF1, SLIT2 and ROBO1 in triple negative breast carcinomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 656. doi:10.1158/1538-7445.AM2013-656
Abstract 66: Pharmacologic tobacco cessation agents and response to radiotherapy.
Authors: Sobus Samantha L. , Romano Michelle A. , Mackowiak Janelle , Pundt Krista , Warren Graham W.
Published: August 2014

Purpose: Tobacco use is associated with poor therapeutic outcomes in cancer patients in part due to decreased therapeutic response to conventional cancer treatment such as chemotherapy and/or radiotherapy (RT). Tobacco cessation is warranted to improve cancer treatment outcomes, but preclinical data suggest that nicotine may decrease the biologic effectiveness of cancer treatment. The purpose of this study is to evaluate if nicotinic acetylcholine receptor (nAChR) and non-nAChR based tobacco cessation pharmacotherapy agents alter response to radiotherapy.

Methods: Human lung cancer (A549, H460) and oropharyngeal cancer (FaDu) cell lines were treated with nicotine, a7-nAChR agonists, varenicline, or bupropion alone or in combination with RT. Mice xenografts were used to assess the effect of nicotine administration during RT. Cell survival and downstream activation of radioprotective pathways (Akt, ERK, mTOR, MMP, and hypoxia inducible factor 1-alpha [HIF-1a]) were analyzed.

Results: Nicotine administration increased survival in all cell lines. Similarly, administration of the selective a7-nAChR agonists LY 2087101 and PHA 54613 was also found to increase cell survival with RT. Nicotine, LY 2087101, and PHA 54613 were all found to activate p-ERK 1/2, p-AKT (Ser-473), mTOR (S2448), and hypoxia inducible factor 1-alpha (HIF-1a) in a dose and time dependent manner. Tumor xenografts receiving nicotine administration during RT showed evidence of increased tumor regrowth in comparison non-nicotine treated controls. Treatment with varenicline also increased cell survival following RT at doses lower than with nicotine treatment; however, treatment with bupropion (an FDA approved non-nAChR based tobacco cessation agent) did not increase cell survival and exhibited a moderate cytotoxic effect following RT at high doses. Dose escalated bupropion did not increase activation of radioprotective pathways.

Conclusions: These are the first data to suggest that bupropion may be an ideal FDA approved tobacco cessation agent in cancer patients to prevent potential biologic changes in therapeutic response associated with nAChR based cessation strategies.

Citation Format: Samantha L. Sobus, Michelle A. Romano, Janelle Mackowiak, Krista Pundt, Graham W. Warren. Pharmacologic tobacco cessation agents and response to radiotherapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 66. doi:10.1158/1538-7445.AM2013-66
Abstract 666: Validation of nucleolar protein 4 as a novel methylated tumor suppressor gene in head and neck cancer.
Authors: Demokan Semra , Chuang Alice Y. , Pattani Kavita M. , Sidransky David , Koch Wayne , Califano Joseph A.
Published: August 2014

Purpose: Methylation of CpG islands in the promoter region of genes acts as a significant mechanism of epigenetic gene silencing in head and neck cancer. In this study, we assessed the utility of epigenetic alterations of a panel of twelve genes (NOL4, IRX1, SLC5A8, LRRC3B, FUSSEL18, EBF3, GBX2, HMX2, SEPT9, ALX3, SOCS3, LHX6) previously reported.

Experimental Design: In this study we investigated methylation of the 12 gene promoters via a candidate gene approach. After the initial screening of methylated CpG islands on the promoter regions by bisulfite sequencing using salivary rinse samples, only two genes had methylated CpG dinucleotides on their promoter regions in tumor samples and absence of methylated CpGs were found in normal salivary rinse samples after bisulfite modification and bisulfite sequencing. We then performed quantitative methylation specific PCR (Q-MSP) on 16 salivary rinse and 14 normal mucosal samples from healthy people and 33 HNSCC tumor samples for two genes selected.

Results: After validation with QMSP, one gene, nucleolar protein 4 (NOL4), was highly methylated (91%) in tumor samples and unmethylated in normal salivary rinse samples and minimally methylated in normal mucosal samples demonstrating cancer specific methylation in HNSCC tissues. Although iroquois homeobox 1 (IRX1) gene was observed as methylated in normal mucosal and salivary rinse samples, indeed, the methylation values of these normal samples were very low (less than 10%).

Conclusions: We identified NOL4, as a highly specific promoter methylated gene was associated with HNSCC. IRX1 which may have a potential as a biomarker for HNSCC and should also be assessed in a larger cohort.

Citation Format: Semra Demokan, Alice Y. Chuang, Kavita M. Pattani, David Sidransky, Wayne Koch, Joseph A. Califano. Validation of nucleolar protein 4 as a novel methylated tumor suppressor gene in head and neck cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 666. doi:10.1158/1538-7445.AM2013-666
Abstract 700: Gene expression profiling of cyclin-dependent kinase (CDK) inhibition in cancer cells.
Authors: Ottaviani Silvia , Delaney Sean , Patel Hetal , Periyasamy Manikandan , Bondke Alexander , Slafer Brian , Starkey Richard , Kroll Sebastian H.B. , Fuchter Matthew J. , Barrett Anthony G.M. , Coombes R. Charles , Ali Simak
Published: August 2014

Cyclin-dependent kinases (CDKs) form complexes with partner cyclins to regulate cell cycle progression and transcription. CDK1, CDK2, CDK4 and CDK6 all regulate cell cycle progression and CDK8 and CDK9 are regulators of transcription. CDK7 is a unique member of this family because it can regulate the cell cycle CDKs as part of the CDK-activating kinase (CAK) and also transcription in conjunction with the TFIIH transcription factor by phosphorylating the COOH-terminal domain of RNA polymerase II (Pol II CTD). A hallmark of cancer is deregulated cell cycle progression and many cancers have abnormal CDK activity which makes CDKs a good target for cancer therapy. Here we show BS-194, a selective and potent CDK inhibitor, which inhibits CDK2, CDK1, CDK5, CDK7 and CDK9 (IC50 = 3, 30, 30, 250 and 90nmol/L, respectively). In vitro cell based assays have shown that BS-194 inhibits the phosphorylation of CDK substrates, Rb and RNA Poll II, down regulation of cyclins A, E and D1 and cell cycle arrest in the S and G2/M phases. BS-194 also has potent anti-proliferative activity in 60 cancer cell lines tested (mean GI50 = 28nmol/L). BS-194 is orally bioavailable and shows potent in vivo tumour growth inhibition of xenograft models of MCF7 and HCT116 cells.

We have used BS-194 as a potent inhibitor of CDK activity in HCT116 cells and performed gene microarray analysis to determine novel pathways and identify novel biomarkers associated with CDK inhibition. In summary, the gene microarray has identified genes differentially expressed in response to BS-194 treatment that are primarily involved in cell cycle, apoptosis and transcription pathways.

Citation Format: Silvia Ottaviani, Sean Delaney, Hetal Patel, Manikandan Periyasamy, Alexander Bondke, Brian Slafer, Richard Starkey, Sebastian H.B. Kroll, Matthew J. Fuchter, Anthony G.M. Barrett, R. Charles Coombes, Simak Ali. Gene expression profiling of cyclin-dependent kinase (CDK) inhibition in cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 700. doi:10.1158/1538-7445.AM2013-700
Abstract 702: Chinese Medicine formulation LQ inhibits FUCCI-expressing HeLa cells at the G0/G1 phase in 2D and 3D culture and invasion with Gelfoam® 3D culture.
Authors: Zhang Lei , Wu Chengyu , Yano Shuya , Hoffman Robert M.
Published: August 2014

Traditional Chinese Medicine formula LQ has previously demonstrated anti-tumor and anti-metastasis efficacy. Here, we used fluorescence ubiquitination-based cell cycle indicator (FUCCI) to monitor cell cycle arrest after LQ treatment. FUCCI-HeLa cells were cultured in two dimensional (2D) monolayer, 3D Matrigel®, and 3D Gelform®. Changes of cell cycle status after 24 hours LQ treatment (90mg/ml) were observed using the Olympus FV1000 confocal imaging system. Paclitaxel (Taxol) was used as the positive control which induced a G2/M block of cell cycle. LQ blocked FUCCI-HeLa cells in the G0/G1 phase of the cell cycle in all cultures. In monolayer culture, the control had approximately 45% of the cells in G2/M phase. In contrast, the LQ-treated cells were mostly in the G0/G1 phase (>90%). In 3D dimensional culture (Matrigel®), cancer cells formed spheres. The colonies in control group had 40% of the cells in G2/M phase, but only 15% in LQ-treated culture. The cancer cells at the surface of the sphere were mostly in G2/M phase, and the cancer cells in the center of colonies were in G0/G1 phase. In 3D Gelform® culture, cells grew along the structures of the Gelform®. The control culture had approximately 45% of cells in G2/M phase. In contrast, the cells in LQ-treated culture were mostly in G0/G1 phase (>80%). The cells in control group migrated to 250∼300 μm deep in the Gelfoam®, but only 150∼200 μm deep in LQ-treated culture. FUCCI-HeLa cells in different culture systems demonstrated the efficacy of LQ on the cell cycle and migration.

Citation Format: Lei Zhang, Chengyu Wu, Shuya Yano, Robert M. Hoffman. Chinese Medicine formulation LQ inhibits FUCCI-expressing HeLa cells at the G0/G1 phase in 2D and 3D culture and invasion with Gelfoam® 3D culture. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 702. doi:10.1158/1538-7445.AM2013-702
Abstract 723: Anticancer ERK kinase inhibitor screening with molecular imaging.
Authors: Zhou Lanlan , Zhang Shengliang , Dicker David T. , Rehemtulla Alnawaz , El-Deiry Wafik S.
Published: August 2014

Extracellular signal-regulated kinase (ERK, also known as the 42-/44-kDa MAPK) signaling pathway controls cell growth, differentiation and survival. ERK is activated by dual phosphorylation on tyrosine and threonine residues (pERK) as a result of genetic alterations in key components of the ERK pathway or upstream activators, or in response to signals from a variety of cell surface receptors. This pro-survival pathway is often upregulated in tumors and is a target for cancer therapy. To screen for ERK inhibitors in small molecule library, an ERK reporter molecule was used to determine ERK kinase activity by measuring bioluminescence with a Xenogen IVIS Optical Imaging System. ERK kinase inhibition was confirmed with Western Blot for pERK to validate ERK reporter. Drug-like small molecule compound libraries NCI Approved Oncology Drug Set (total 2 Plates, 101 compounds) and NCI Diversity Set II (total 18 Plates, 1364 compounds) were screened with an ERK reporter expressing cell line BxPC3-MBP by using bioluminescent assay at different time points and different dosages. Then top 15 candidates were further tested for cell toxicity IC50, ERK kinase inhibition, EGFR kinase inhibition, and combinational treatment response with chemotherapy agents in normal cell MRC-5, colorectal cancer cell SW480, and non-small cell lung cancer cell H460 and Calu6. ERK kinase inhibitor candidate number 6 was found to inhibit ERK kinase activity in a dose-dependent manner and have relatively lower toxicity to normal cells than cancer cells. Clinical trials of Sorafenib (an established small molecular inhibitor of several Tyrosine protein kinases and Raf kinases) combined with Cisplatin and Etoposide on small cell lung cancer and non-small cell lung cancer showed not favorable so Etoposide was compared with its similar structure Teniposide in our combination treatment. Combinational treatments were tested here are ERK kinase inhibitor candidate/Sorafenib combined with Cisplatin and Etoposide/Teniposide. Our results demonstrated that comparing with Sorafenib, ERK kinase inhibitor candidate number 6 was more efficient in killing non-small cell lung cancer cells in combinational treatment with Teniposide and Cisplatin. This ERK kinase inhibitor candidate has shown considerable promise for further in vivo study and clinical development.

Citation Format: Lanlan Zhou, Shengliang Zhang, David T. Dicker, Alnawaz Rehemtulla, Wafik S. El-Deiry. Anticancer ERK kinase inhibitor screening with molecular imaging. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 723. doi:10.1158/1538-7445.AM2013-723
Abstract 73: Preclinical evaluation of chemoradiation with cetuximab HPV-positive head and neck cancer suggests significant radiosensitization.
Authors: Smith Molly A. , Blitzer Grace C. , Torres Alexandra D. , Armstrong Eric A. , Lambert Paul F. , Harari Paul M. , Kimple Randall J.
Published: August 2014

PURPOSE: Whether cetuximab, a monoclonal antibody to the epidermal growth factor receptor, should be combined with radiation in patients with human papillomavirus (HPV) positive locally advanced head and neck cancer (HNC) is not known. Currently available data are inadequate to address this important therapeutic question. We sought to examine the ability of cetuximab to act as a radiosensitizer and to examine the mechanism of action of this combination in a model system of HPV+ HNC.

METHODS: Four cell lines (UD-SCC2, UM-SCC47, UPCI-SCC90, 93-VU-147T) validated for HPV expression were used for all assays. Subcutaneous flank xenografts were performed in Hsd:athymic Nude-Foxn1nu female mice treated with intraperitoneal cetuximab (0.2mg/mouse) or flank radiation (2 Gy/fraction) delivered twice weekly for 2 weeks. Time to tumor quadrupling from baseline was assessed by Kaplan-Meier method. Flow cytometry was used to assess apoptosis by Annexin V staining and cell cycle distribution by propidium iodide staining.

RESULTS: While there was minimal in vitro radiosensitization after treatment with cetuximab (similar to that seen in HPV-negative HNC), cetuximab + radiation resulted in a significant increase in time to tumor quadrupling compared to cetuximab alone (p=0.001) or radiation alone (p<0.0001) in HPV+ HNC xenografts. Increased apoptosis was seen in cetuximab and radiation treated cells. Analysis of cell cycle distribution 24 hours after drug treatment showed that cetuximab induced a modest G1 cell cycle arrest when given prior to radiation, but that 24 hours after combined therapy, a significant G2 arrest was present.

CONCLUSIONS: Using HPV+ HNC cell lines, radiation and cetuximab appear to be beneficial compared to either treatment alone. This combination, currently undergoing study in randomized clinical trial is being further evaluated in a direct from patient tumorgraft model of HPV+ HNC to further understand predictors of response.

Citation Format: Molly A. Smith, Grace C. Blitzer, Alexandra D. Torres, Eric A. Armstrong, Paul F. Lambert, Paul M. Harari, Randall J. Kimple. Preclinical evaluation of chemoradiation with cetuximab HPV-positive head and neck cancer suggests significant radiosensitization. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 73. doi:10.1158/1538-7445.AM2013-73
Abstract 731: In vivo fluorescence labeling of gastrointestinal stromal tumor (GIST) .
Authors: Metildi Cristina A. , Tang Chih-Min , Kaushal Sharmeela , Leonard Stephanie Y. , Magistri Paolo , Hoffman Robert M. , Bouvet Michael , Sicklick Jason K.
Published: August 2014

Gastrointestinal stromal tumor (GIST), the most common sarcoma, metastasizes hematogenously and peritoneally. Tumor-free margins and complete cytoreduction of metastatic disease are critical for decreasing recurrence. Since most GISTs overexpress KIT (c-KIT, CD117), we hypothesized that fluorescently labeled anti-KIT antibodies can improve detection of GIST. KIT K641E+/− transgenic mice that spontaneously develop cecal GIST, and wild-type C57BL/6 mice were used in this study. Wistar rat anti-mouse monoclonal c-KIT antibody was labeled with the AlexaFluor 488. The conjugated anti-KIT-Alexa 488 antibody (50-100 μg) was delivered via tail vein at 24 hours prior to standard staging laparoscopy. A pediatric laparoscopic grasper was inserted in the left lower quadrant for bowel mobilization in order to visualize the cecum. All 4 quadrants of the peritoneal cavity were examined systematically under both fluorescence and bright light. Post laparoscopy, mice were sacrificed and their abdomens exposed for Olympus OV-100 imaging. All tumors were resected and confirmed by H&E. We divided the mice into 4 groups. KIT K641E+/− mice received anti-KIT antibody (N=4) or control antibody (N=3). Wild-type mice received the same antibodies (N=3 per group). Fluorescence laparoscopy demonstrated a high tumor signal-to-background noise ratio. Upon whole body imaging used to detect gross tumors, there were 2 false positive and 0 false negative results, leading to an accuracy of 92%. The sensitivity, specificity, positive and negative predictive values were: 100%, 87%, 85%, and 100%, respectively. This method has several translatable applications: 1) identification of peritoneal metastases; 2) following disease response; 3) non-radioactive imaging; 4) endoscopic differentiation of gastric GISTs from leiomyomas or schwannomas; 5) laparoscopic staging; and 6) assessment of margin status.

Citation Format: Cristina A. Metildi, Chih-Min Tang, Sharmeela Kaushal, Stephanie Y. Leonard, Paolo Magistri, Robert M. Hoffman, Michael Bouvet, Jason K. Sicklick. In vivo fluorescence labeling of gastrointestinal stromal tumor (GIST) . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 731. doi:10.1158/1538-7445.AM2013-731
Abstract 737: Multispectral Optoacoustic Tomography (MSOT) imaging and quantification of apoptosis in vivo.
Authors: Driessen Wouter H. , Burton Neal C. , Sardella Thomas , Morscher Stefan , Razansky Daniel , Ntziachristos Vasilis
Published: August 2014

Apoptosis is an important mechanism in cellular homeostasis and imbalances in the apoptotic process are associated with various disease states. An important example is the acquired ability of cancer cells to resist their own programmed cell death and therefore it is the aim of many tumor therapies to either reestablish pro-apoptotic signaling pathways or induce apoptosis through activation of existing mechanisms within the cell. Therefore, visualizing and quantifying the apoptotic process in vivo has great value in monitoring therapy response, diagnosis and staging disease.

By being able to detect subtle changes in perfusion and tissue oxygenation, the small animal Multispectral Optoacoustic Tomography (MSOT) scanner offers unprecedented performance in cross-sectional imaging of tumor heterogeneity as well as a powerful capacity to simultaneously image molecular processes such as apoptosis by utilizing novel molecular probes. In this study, apoptotic regions within a mouse mammary tumor where visualized using a dye-conjugated caspase probe and compared to the hypoxia status of each tumor region.

The temporal resolution of the MSOT small animal scanner (generation of multispectral cross-sectional data in less than 1 second) allows for the dynamic imaging of targeted and control probe simultaneously. To illustrate this point apoptosis probe was co-injected with a control dye with similar physical and chemical properties, but a different absorbance maximum after which the tumor region was imaged over 1 hour with multi-spectral image acquisition. Next, mice were imaged by MSOT immediately before and 24 hours post treatment with the chemotherapeutic Doxorubicin and the induction of apoptosis was visualized and quantified. Doxorubicin treatment led to a significant increase in signal resulting from the DyLight 747-conjugated apoptosis probe. In summary, MSOT can be used to determine the extent of apoptosis in tumors in vivo and thus has great value in monitoring therapy response, diagnosis and staging disease.

Citation Format: Wouter H. Driessen, Neal C. Burton, Thomas Sardella, Stefan Morscher, Daniel Razansky, Vasilis Ntziachristos. Multispectral Optoacoustic Tomography (MSOT) imaging and quantification of apoptosis in vivo. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 737. doi:10.1158/1538-7445.AM2013-737