bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1750: Reduced xpression of Reelin (RELN) gene is associated with high recurrence rate of hepatocellular carcinoma
Authors: Okamura Yukiyasu , Nomoto Shuji , Kanda Mitsuro , Hayashi Masamichi , Fujii Tsutomu , Sugimito Hiroyuki , Takeda Shin , Nakao Akimasa
Published: February 2011

Background and Aims: Hepatocellular carcinoma (HCC) is one of the world's top five causes of cancer-related deaths and has limited treatment options. HCC is one of the few cancers for which the etiology of the underlying disease is known in most cases. However, the molecular pathogenesis of HCC has not been fully elucidated. Recently described genome-wide approaches robustly detect many candidate genes that are regulated in HCC. Here we attempt to identify the novel genes with altered expressions related to clinical factors using a double-combination array, which contains an expression array and a single nucleotide polymorphism (SNP) array. Moreover, we analyzed the identified gene, using quantitative reverse transcription polymerase chain reaction (RT-PCR) and methylation-specific PCR.

Samples and Methods: A 68-year-old woman with chronic hepatitis C was diagnosed with HCC and underwent liver resection. On the same HCC sample, we performed double-combination array analysis. We consequently analyzed the identified gene, using quantitative RT-PCR, methylation-specific PCR, and un-methylated specific PCR in five HCC cell lines, together with 48 pairs of HCC tissues (HTs) and adjacent non-tumorous tissues (NTs) obtained from patients who underwent hepatectomy. The results were compared with the clinico-pathological data.

Results: There was decreased expression of the Reelin (RELN) gene in HT at the level of −3.0. We then used the SNP-Chip array to confirm that the copy number of chromosome 7 where RELN is located did not show any abnormalities. These results suggest that RELN expression was reduced without LOH or deletion. We then examined the sequence of the promoter region of RELN gene and found many CpG islands. Therefore, we hypothesized that hypermethylation of the CpG islands was the mechanism for its decreased expression in HT. Thirty-four of 48 (79.2%) samples had decreased RELN expression level in HTs and the expression level of HTs was clearly reduced compared with that of NTs (P < 0.002). We found that 18 of 48 (37.5%) HTs had promoter hypermethylation of the RELN gene. In regard to clinical data, the patients with reduced expression in HTs have a high recurrence rate (P = 0.028).

Conclusion. The present study suggests that our method is able to detect novel genes with altered expression and that RELN is a novel gene associated with recurrence of HCC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1750.
Abstract 1759: MK-1775, a novel Wee1 kinase inhibitor, radiosensitizes p53-defective non-small cell lung cancer (NSCLC) cells
Authors: Bridges Kathleen , Hirai Hiroshi , Buser Carolyn , Meyn Raymond E.
Published: February 2011

Wee1 is a tyrosine kinase involved in the S>G2 and G2>M cell cycle transitions. Following DNA damage, Wee1 is phosphorylated by upstream kinases and stabilized. Stabilized Wee1, in turn, phosphorylates and inhibits CDC2, preventing the cells from entering mitosis before repairing their damaged DNA. Abrogation of Wee1 activity, either by decreasing expression using siRNA, or by inhibiting protein activity with selective small molecule inhibitors, sensitizes tumor cells to DNA-damaging agents. This sensitization preferentially takes place in a p53 null or mutant context. We have investigated the ability of a novel, small molecule inhibitor of Wee1, MK-1775, to radiosensitize NSCLC cells treated in vitro. Four NSCLC cell lines were used in the study: A549 and H460 with wild-type p53 status and H1299 and Calu6 with null or mutant p53 status, respectively. A 24 hr pre-irradiation treatment with 200 nM MK-1775 suppressed phosphorylation of CDC2 in both A549 and H1299 cells, however, this treatment did not produce a radiosensitizing effect in either line. An 18 hr post-irradiation treatment slightly radiosensitized the H1299 line but not the A549 line. The optimal treatment of a 1 hr pre-irradiation followed by an 18 hr post-irradiation treatment substantially radiosensitized the H1299 and Calu6 lines, yielding dose-enhancement factors (DEFs) at 10% survival of 1.39 and 1.47 respectively, but did not sensitize the A549 or H460 cells, DEFs of 1.0. To determine the mechanism responsible for the radiosensitizing effect of MK-1775, cells were treated with nocodazole following a dose of 4 Gy and analyzed for phospho-histone H3 as an indication of mitotic index after 4 hrs. Unirradiated cells accumulated in mitosis during the 4 hrs in nocodazole and this accumulation was substantially enhanced by MK-1775 in both p53 wild type A549 and p53 null H1299 cells suggesting that Wee1 inhibition accelerates unirradiated cells into mitosis prematurely. Neither A549 nor H1299 cells, irradiated with 4 Gy, accumulated in mitosis during the 4 hr nocodazole treatment in the absence of MK-1775 indicating a robust G2 block for both lines. However, H1299 cells irradiated with 4 Gy and treated with MK-1775 accumulated in mitosis to a level higher than the unirradiated cells treated with MK-1775 alone. This enhanced entry of irradiated cells into mitosis was not evident to nearly the same degree in the A549 cells. Thus, we conclude that the Wee1 inhibitor, MK-1775, accelerates cells harboring DNA-damage prematurely into mitosis in a p53-dependent manner correlating with its radiosensitizing effects.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1759.
Abstract 1761: Class I HDAC expression patterns in gastroenteropancreatic neuroendocrine tumors
Authors: Darb-Esfahani Silvia , Rieger Anja , Pavel Marianne , Kasajima Atsuko , Sasano Hironobu , Dietel Manfred , Denkert Carsten , Wiedenmann Bertram , Weichert Wilko
Published: February 2011

Aims Inhibition of histone deacetylases (HDACs) has been shown to have antiproliferative and proapoptotic effects on gastrointestinal neuroendocrine tumor cells in vitro. While several HDAC inhibitors have entered clinical trials as antineoplastic agents in various tumor entities, information on the expression of HDACs in gastroenteropancreatic neuroendocrine tumors (GEP-NET) in vivo is still lacking.

Methods We investigated expression of class I HDAC1, HDAC2 and HDAC3 in a large cohort of neuroendocrine tumors by immunohistochemistry and correlated our findings with clinicopathologic variables, proliferative activity and patient prognosis.

Results While 58 (59.2%) out of 98 neuroendocrine foregut and midgut tumors did not display any nuclear expression of HDAC1, HDAC2 or HDAC3, 38 (38.8%) were partially positive, i.e. showed nuclear staining for at least one of the three HDAC isoforms. Only 2 (2.0%) tumors were positive for all of the three HDACs.

Nuclear expression of HDAC isoforms correlated strongly (p<0.006) with each other and also correlated with proliferative capacity as determined by expression of Ki67 (p=0.003). Consequently, expression of HDAC1 in foregut tumors was correlated with grading (p=0.009) and a similar tendency was seen for HDAC2 (p=0.077). No correlation was seen between HDAC expression in primary tumors and tumor stage, nodal status or state of metastasis. However, HDAC expression was lower in nodal foregut and midgut lesions when compared to the corresponding primary tumors (p<0.023). HDAC expression was not associated with disease specific survival in stage IV midgut tumors.

Conclusion Our observation of a tight association of HDAC positivity and proliferative activity in GEP-NETs mirror the fact that overexpression of HDACs promote cancer cell growth in these neoplasms in vivo. We additionaly hypothesize that differences in HDAC expression might predict response to HDAC inhibitors which should be taken into account when future clinical trials with these substances are being planned.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1761.
Abstract 1764: Horizontal and vertical signal pathway inhibition in human tumor primary culture micro-spheroids
Authors: Nagourney Robert Alan , Bernard Paula J. , Francisco Federico R. , Evans Steven S.
Published: February 2011

Introduction: Signal transduction pathways are targets for small molecule tyrosine and serine-threonine kinase inhibitors. Redundancy and cross talk between pathways can complicate the application of genomic signatures but proteomic and functional platforms may more closely approximate tumor phenotypes. We used EVA/PCD (ex vivo analysis of programmed cell death), a functional platform measuring both apoptotic & non-apoptotic events to examine the PI3K and EGFr pathways in human 1° culture micro-spheroids. Methods: 337 tumor specimens provided from the OR were mechanically and enzymatically disaggregated and exposed to EGFr (Gefitinib), AKT (B-15), PI3K (LY294002) & mTOR (Rapamycin) inhibitors with LC50 values interpolated from 5-point dose response curves. Synergy was assessed by median effect and correlation coefficients by Pearson Moment. Results: Parallel Pearson Moment analyses in 51 specimens by 2-tailed T, revealed LY vs. B15 (NS); LY vs. Rapa (P<0.02); B15 vs. Rapa (P<0.0025); Rapa vs. Gefitinib (P <0.001). Synergy analyses for Rapa & Gefitinib revealed synergy in 29%, additivity in 41%, sub-additivity in 13% and antagonism in 17%. Exploratory synergy analyses that combined Gefitinib & LY and Gefitinib & B-15, favored the Gefitinib& B-15 combination with 100% synergy in this small series. Conclusions: EVA/PCD analyses provide insights into cellular response to targeted therapies, alone and in combination. Correlative analyses identify points of commonality and/or disparity in downstream signaling that can be exploited in drug development and therapy. Synergy analyses reveal potentially important drug combinations. The EVA/PCD human tumor micro-spheroid platform provides a functional tool capable of streamlining drug development and cancer therapy. Supported by The Vanguard Cancer Foundation and the Nagourney Institute.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1764.
Abstract 1765: Preclinical optimization of the Chk1/2 inhibitor AZD7762 in combination with gemcitabine in pancreatic cancer
Authors: Parsels Leslie A. , Morgan Meredith A. , Tanska Daria M. , Parsels Joshua D. , Davis Mary A. , Hassan Maria C. , Arumugarajah Sankari , Hylander-Gans Linda , Morosini Deborah , Zhao Lili , Simeone Diane M. , Canman Christine E. , Normolle Daniel P. , Zabludoff Sonya D. , Lawrence Theodore S. , Maybaum Jonathan
Published: February 2011

Pancreatic cancer remains among the most lethal cancers with an overall 5-year survival of less than 5%. Chk1 inhibition has emerged as a promising approach to sensitize pancreatic cancer to gemcitabine (Gem), the current standard therapy for both local control and metastatic disease. Here, we characterized the ability of AZD7762, a small molecule Chk1/2 inhibitor currently in Phase I trials, to sensitize pancreatic cancer cells to Gem both in vitro and in vivo.

We first evaluated the in vitro schedule-dependence of AZD7762-mediated Gem sensitization by clonogenic assay. We found that AZD7762 effectively sensitized pancreatic cancer cell lines to Gem-induced loss of clonogenicity, with shifts in IC50 from 3- to 38-fold, compared to cells treated with Gem alone. At low concentrations of Gem (≤IC50), AZD7762 was most effective when present during Gem treatment. At higher concentrations of Gem, however, giving AZD7762 24 h after Gem resulted in the greatest sensitization. We next assessed the molecular mechanisms of AZD7762-mediated chemosensitization in one of these cell lines, MiaPaCa2, by immunoblot, flow cytometry and immunofluorescent staining. Gemcitabine sensitization was associated with inhibition of Chk1, rather than Chk2 and abrogation of the S-M and G2-M checkpoints. Despite checkpoint abrogation, we found that Gem/AZD7762-treated cells were unable to exit S-phase and, over time, AZD7762 inhibited DNA synthesis, as measured by BrdUrd incorporation. This inhibition was associated with pan γ-H2AX staining in Gem-treated cells, in addition to disruption of Rad51 foci and inhibition of homologous recombination repair activity.

We next evaluated the effects of AZD7762 in two different human pancreatic cancer xenograft models; MiaPaCa2, and a patient-derived early passage pancreatic cancer xenograft, EPX-J. AZD7762 had anti-tumor activity both as a single agent, and in combination with Gem in both MiaPaCa2 and EPX-J, with average times to tumor doubling of 37 and 49 days, respectively, in tumors treated with both Gem/AZD7762, compared to 18 and 29 days for tumors treated with Gem alone (*p≤0.05). Chk1 inhibition, as determined by S296- and S345-Chk1 phosphorylation status, correlated with in vivo response. Finally, we found increased S345-Chk1 phosphorylation in normal tissues from mice treated with Gem/AZD7762, suggesting this may be a useful surrogate marker for AZD7762 activity in vivo.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1765.
Abstract 1770: Overexpression of fibroblast growth factor receptor 2 (FGFR2) in gastric cancer and basis for future FGFR2-targeted cancer therapy
Authors: Yeh Kun-Huei , Shun Chia-Tung , Kao Chiu-Hua , Lin Yu-Lin , Shen Ying-Chun , Cheng Ann-Lii
Published: February 2011

Introduction: Fibroblast growth factors (FGFs) are potent mitogens and inducers of angiogenesis. FGF signalling pathway is closely associated with cancer development and progression. Fibroblast growth factor receptor 2 (FGFR2) gene, located at human chromosome 10q26, encodes FGFR2b and FGFR2c isoforms functioning as distinct FGF receptors. Gene amplification or missense mutation of FGFR2 occurs in gastric cancer (GC). Genetic alterations of FGFR2 may induce aberrant activation of FGFR2 signalling pathway, thereafter induce proliferation and survival of GC cells. FGFR2 plays an important role in gastric carcinogenesis.

Patients and Methods: A total of 33 gastric cancer tissues with formalin-fixed, paraffin-embedded tumor slides were investigated by immunohistochemical staining with primary antibody against FGFR2. By Lauren's classification, there are 9 diffuse-type, 23 intestinal-type, and 1 mixed-type GC patients, respectively.

Results: The FGFR2 immunohistochemical staining was classified as 0, 1+, 2+, and 3+ according to percentage of immunoreactive positive-staining FGFR2 cells in <10%, 10-25%, 25-50%, and >50% tumor cells, respectively. The relationship between the FGFR2 expression and the clinicopathological characteristics were analyzed. Among 33 GC patients, 13 patients (39.4%; 23-57%, 95% C.I.) had FGFR2 expression (1+, 2+, and 3+), and 11 patients (33.3%; 17-52%, 95% C.I.) had FGFR2 overexpression (2+ and 3+), respectively. FGFR2 expression was not correlated with diffuse-type versus intestinal-type GC patients (p=0.427, Fisher exact test).

Conclusions: Overexpression of FGFR2 in gastric cancer forms the basis of the rational clinical application of future FGFR2-targeted cancer therapy for treatment of advanced GC.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1770.
Abstract 1771: Therapeutic management of pancreatic cancer using a novel drug, Minnelide
Authors: Chugh Rohit , Patil Satish , Sangwan Veena , Vickers Selwyn , Georg Gunda , Saluja Ashok K.
Published: February 2011

Background: Pancreatic cancer, the fourth-leading cause of cancer-related deaths in the United States, is one of the most malignant and aggressive forms of cancer, with a 5-year survival of <5%. We have previously shown that triptolide, a diterpenoid isolated from Tripterygium wilfordii, inhibits HSP70 expression leading to cell death in pancreatic cancer cells and reduces the growth as well as loco-regional spread of pancreatic tumors in an orthotopic model. The drawback of the use of triptolide in the clinic is its insolubility in water, requiring the presence of organic solvents. We therefore developed a prodrug of triptolide named Minnelide which is water soluble and converts to the parent compound both in vitro and in vivo.

Aim: To evaluate the efficacy of Minnelide as a potential therapeutic agent for pancreatic cancer.

Methods: Pancreatic cancer cells of varying metastatic potential (MIA PaCa-2/S2-VP10/S2-013) were incubated with different concentrations of Minnelide (100-200 nM) or vehicle (control). Cell viability was measured using an MTT-based assay, and apoptosis assessed using annexin V staining and caspase 3/9 activation at different time intervals. HSP70 expression was measured in lysates from Minnelide-treated cells by western blot. In vivo efficacy of the drug was measured by implanting tumor cells into the pancreas of nude mice which were then treated with either vehicle or Minnelide for different periods of time, and at different stages of tumor formation.

Results: Minnelide decreased the cell viability of the three different human pancreatic cancer cell lines tested in a dose- and time-dependent manner. Furthermore, cell death was associated with annexin V positivity and activation of caspase 3/9. Minnelide repressed HSP70 protein expression at 100nM, supporting our previous results which show that HSP70 plays an important role in tumor progression. In the in vivo orthotopic mouse model, Minnelide dramatically decreased tumor size, loco-regional spread of tumor and extended survival, both in the early (7 days post-inoculation) and late (28 days post-inoculation) stages of pancreatic cancer. Importantly, toxic studies have shown that Minnelide at the concentrations required for tumor regression is not toxic to the animal.

Conclusion: Minnelide is a potent anti-cancer agent, both in vitro and in vivo, causing cell death via apoptosis and tumor regression in vivo. It holds great promise as a novel single-agent therapeutic strategy against pancreatic cancer. We are currently in the process of initiating Phase I clinical trials for this drug.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1771.
Abstract 1772: Calcium activated nucleotidase 1 (CANT1) promotes progression of prostate carcinomas
Authors: Gerhardt Josefine , Steinbrech Corinna , Fritzsche Florian , Tischler Verena , Müntener Michael , Sulser Tullio , Stephan Carsten , Jung Klaus , Moch Holger , Kristiansen Glen
Published: February 2011

Aims:

Previously, we identified Calcium-activated nucleotidase 1 (CANT1) mRNA to be overexpressed in human prostate cancer, however, so far no involvement of CANT1 in the progression of human tumours was reported. Therefore the protein expression level of CANT1 in prostate tumours was determined, followed by the elucidation of a functional implication of CANT1 overexpression in prostate cancer progression.

Methods:

Two tissue microarrays including 1100 prostate samples from two different institutions were constructed to examine the protein expression level of CANT1. The role of CANT1 in tumour progression was assessed in two prostate cancer cell lines in an RNA interference based approach. Cell number and DNA synthesis rate were determined by in vitro assays to measure cell proliferation, further cell migration was studied in transwell chamber assays.

Results:

A recurrent overexpression of CANT1 protein in human prostate cancers was confirmed on the tissue microarrays. Functionally, CANT1 knockdown induced a reduction of LNCaP as well as PC-3 cell number, which was caused by a diminished DNA synthesis rate. Moreover, a considerable decrease of cell migration towards a fibronectin gradient was observed in both cell lines upon CANT1 knockdown. This decreased cell mobility was also accompanied by morphological cell changes.

Conclusions:

Two cellular processes that are directly linked to tumour progression are markedly impaired upon CANT1 knockdown in prostate cancer cell lines, demonstrating for the first time a tumourbiological relevance of CANT1 expression. In future experiments the mechanism how CANT1 exerts its effects on proliferation and migration will be elucidated as well as the pathways that lead to CANT1 overexpression in prostate cancer. This will contribute to a better understanding of prostate carcinogenesis and to the evaluation of the potential of CANT1 as a therapeutic target.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1772.
Abstract 1773: The addition of Reolysin, an oncolytic reovirus, to irinotecan shows synergistic anticancer activity in colorectal cancer cell lines
Authors: Seetharam Raviraja N. , Coffey Matthew C. , Klampfer Lidija , Mariadason John , Goel Sanjay
Published: February 2011

Recent clinical data suggests that the anti EGFR antibodies, cetuximab and panitumumab, are ineffective in patients with colorectal cancer whose tumors harbor a mutation in the kras oncogene. Currently, for these patients, the only option after failure of front line therapy is irinotecan, and there is no drug that can be combined with it to improve clinical outcomes. We studied in vitro, the combination of Reolysin and irinotecan to develop a novel therapeutic approach for these patients.

Reolysin (reovirus Serotype 3) has been shown to replicate specifically in tumors bearing an activated ras pathway. This specificity coupled with its relatively nonpathogenic nature in humans makes it an attractive anti-cancer therapy candidate. We have performed in vitro studies of Reo (multiplicity of infection 1 to 10), iri (0.5-10 µM), as single agents and in combination in human colorectal cancer cells to study the role of kras mutation.

Reolysin was obtained from Oncolytics Biotech Inc. and irinotecan was obtained commercially. A panel of 8 colorectal cancer cell lines were infected with Reolysin (multiplicity of infection 1 to 10), irinotecan (0.5-10 µM), as a single agent or in combination. The cells were exposed to Reolysin for 6-8 hours and to irinotecan for 72 hours. Viable cells were determined 72 hours post treatment using MTT assay. The effect of Reolysin and irinotecan combination on cell viability was assessed using CalcuSyn software (Biosoft, UK) that generates combined cytotoxic effect by calculating combination indices (CIs). A CI of <1 indicates synergy, 1 denotes additive effect, and >1 denotes antagonism.

We found that Reolysin and irinotecan as single agents were cytotoxic to all colon cell lines studied. Further, when Reolysin was combined with irinotecan, there was evidence of synergistic cytotoxicity in all (HT29, HCT116, DLD, KM12, SKCO-1, SW620, LIM2405) except one (SW948) cell line. There appears to be no association between kras mutation status and synergism. To further explore these findings and understand the mechanistic basis of drug synergism we are studying the effect of Reolysin and irinotecan in isogenic HCT116 (kras mutant) and Hke3 (kras WT) cell lines.

In summary, the combination of Reolysin and irinotecan is synergistic in colorectal cancer cell lines including those with kras mutation and is worthy of exploration in human patients.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1773.
Abstract 1774: Selection for the EGFR T790M gatekeeper resistance mutation may vary among different small molecule EGFR TKIs
Authors: Chmielecki Juliann , Pietanza Maria C. , Kris Mark , Miller Vincent , Stout Thomas , Pao William
Published: February 2011

Mutations within exons encoding the kinase domain of EGFR confer increased sensitivity of non-small cell lung carcinomas to the tyrosine kinase inhibitors (TKIs), gefitinib and erlotinib. Unfortunately, all patients whose disease responds initially to these therapies eventually develop acquired resistance. In ∼50% of cases, resistance is associated with acquisition of a second-site ‘gatekeeper’ mutation within EGFR, T790M. More potent ‘2nd generation’ EGFR inhibitors theoretically able to overcome T790M-mediated resistance have displayed limited clinical success. In preclinical models of acquired resistance, many of these TKIs (HKI-272, BIBW2992, PF00299804) actually select for the emergence of EGFR T790M-harboring clones.

To determine if EGFR TKIs in general lead to the emergence of T790M, we modeled acquired resistance in vitro with the reversible TKI, XL647 (Symphony Evolution, Inc., Rockville, MD), the irreversible EGFR TKI, BIBW2992 (synthesized at MSKCC), and erlotinib. EGFR mutant TKI-sensitive PC-9 cells (lung adenocarcinoma, del E746-A750) were grown with increasing concentrations of XL647 for ∼160 days. Polyclonal resistant (XL-R) cells were derived that proliferated in drug concentrations ∼50-fold higher than the initial IC50. No other secondary mutations within EGFR were detected by direct sequencing, and the T790M mutation was not found using more sensitive mutation detection methods (e.g. SNaPShot; limit of detection ∼5%). By contrast, both BIBW2992- and erlotinib-resistant PC-9 cells (PC-9 BI-R and PC-9 E-R) harbored T790M, as shown by direct sequencing. No resistant cells displayed MET amplification, another known resistance mechanism. Crystal structure data revealed that XL647 binds reversibly within the ATP binding pocket of EGFR T790M with a similar orientation to erlotinib. Therefore, structural differences alone do not readily explain the differential selective pressure for EGFR T790M with gefitinib/erlotinib vs. XL647. Interestingly, PC-9 XL-R cells remained intermediately sensitive to erlotinib. Consistent with the in vitro observations, early clinical trial data revealed that only 1 of 6 patients with EGFR mutant lung cancer harbored T790M after developing acquired resistance to XL647. Furthermore, at least 2 of these patients subsequently derived clinical benefit from single-agent erlotinib. Collectively, these data suggest that the propensity to develop T790M-mediated resistance varies among different TKIs. We are currently exploring why the selective pressure for resistance conferred by XL647 is different from that of other EGFR TKIs.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1774.
Abstract 1775: Growth inhibitory effect of PARP inhibitor olaparib in gastric cancer cells
Authors: Im Seock-Ah , Min Ahrum , Hur Hyung-Seok , Yoon Young-Kwang , Song Sang Hyun , Riches Lucy , Knights Charlotte , Han Sae-Won , Oh Do-Youn , Wombwell Helen , Kim Tae-You , Gu Yi , Lau Alan , Bang Yung-Jue , O'Connor Mark
Published: February 2011

Background Poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP)is the member of a family of enzymes that catalyze the addition of ADP-ribose units to proteins that mediate DNA repair pathways. Olaparib (KU-0059436; AZD2281) is a potent oral inhibitor of PARP currently in clinical development, which has selective antitumor activity in cancers, associated with BRCA1 and BRCA2 mutations. Gastric cancer is the most common cancer and the second leading cause of cancer deaths in Korea. New therapies including molecular targeting agents are eagerly awaited, and treatment strategies to overcome chemoresistance are also needed in the treatment of gastric cancer. Materials and Methods We studied the growth inhibitory effects of Olaparib on gastric cancer (GC) cells using clonogenic survival assays and studied the effect of combination with chemotherapeutic agents using MTS assays. Cell cycle analysis and molecular changes induced by olaparib were also performed. Results Olaparib showed marked growth inhibitory activity against the SNU 601 cell line, with IC50 value of 0.015 μM. In addition, 5 out of 11 GC cells (45%) were sensitive to olaparib with IC50s ≤ 2μM. Olaprib induced G2/M cell cycle arrest and apoptosis in sensitive GC cells. We identified synergistic growth inhibitory effects of Olaparib in combination with clinically relevant cytotoxic agents (5-FU, cisplatin, and oxaliplatin). Furthermore, Olaparib-induced apoptosis was associated with PARP cleavage and caspase-3 activation. Conclusion: Olaparib showed growth inhibitory activity against GC cells as a single agent and acted synergistically with cytotoxic agents. These results provide a rationale for the future clinical trials of olaparib combined with cytotoxic drugs in the treatment of gastric cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1775.
Abstract 1782: Detection of T790M gefitinib resistance mutation in EGFR using the BEAMing method
Authors: Taniguchi Kazuya , Nishitani Kimiyoshi , Okami Jiro , Kodama Ken , Higashiyama Masahiko , Kato Kikuya
Published: February 2011

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), such as gefitinib or erlotinib, are effective therapies for non-small cell lung cancer (NSCLC) patients with primary EGFR mutations. However, most patients who initially respond to treatment with these drugs will develop resistance to EGFR TKIs. The secondary EGFR T790M mutation is reported to correlate with acquired resistance. We examined the T790M mutation in primary tumors with BEAMing (beads, emulsion, amplification, magnetic), which is the most sensitive technique to detect minor mutated alleles. Currently, we can detect point mutation at the level of 1/10,000 using an improved version of the BEAMing. We detected T790M mutant alleles in NSCLCs in 19 out of 270 NSCLCs with primary EGFR mutations. The detection of small fraction of T790M mutant alleles may be useful for predicting resistance of NSCLCs to TKIs.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1782.
Abstract 1784: Erlotinib at a dose of 25 mg daily for non-small cell lung cancers with EGFR mutations
Authors: Yeo Wee-Lee , Riely Gregory J. , Kris Mark G. , Tenen Daniel G. , Pao William , Kobayashi Susumu , Costa Daniel B.
Published: February 2011

Introduction: The tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib are active in non-small-cell lung cancers (NSCLCs) with epidermal growth factor receptor (EGFR) gene mutations. The usual clinical dose of gefitinib (250 mg/day) is only one third of its maximum tolerated dose (MTD), while the dose of erlotinib (150 mg/day) is at its MTD. In NSCLC cell lines both TKIs have similar micromolar (μM) inhibitory concentrations. We explored if erlotinib at 25 mg/day (presumed trough serum concentration similar to gefitinib 250 mg/day at around 0.5 μM) would be active in EGFR mutated NSCLC.

Methods and Patients: To study inhibitory concentrations of gefitinib and erlotinib, we studied EGFR mutated cell lines (HCC827, H3255, PC-9, H1975) exposed to increasing concentrations of these TKIs. Further on, we performed a retrospective evaluation of seven patients with advanced EGFR mutated (exon 19 deletions and L858R) NSCLC that were given erlotinib at 25 mg daily as their first EGFR TKI.

Results: In EGFR mutated cell lines, both gefitinib and erlotinib had median IC50 inhibitory concentrations with similar values and statistical correlation (Pearson r = 0.9999, p < 0.0001). Concordance between the IC50 of gefitinib and erlotinib was confirmed using a separate published dataset of 8 EGFR-mutant NSCLC lines (r = 0.9919, p < 0.0001). These results indicate that both EGFR inhibitors have similar inhibitory concentrations in EGFR mutated cells. Of the 7 patients treated with erlotinib 25 mg/day, 5 (71.5%) had partial radiographic responses. Median progression-free survival (PFS) was 17 months, and individual PFS exceeds 6 months in most patients that have received drug for at least that period. Toxicities were minimal with only 2 (28.5 %) patients having a rash (grade 1) and none (0%) experiencing diarrhea.

Conclusions: In patients with EGFR activating mutations, a dose of erlotinib 25 mg/day leads to impressive response rate and PFS similar to the growing experience with gefitinib 250 mg/day and erlotinib 150 mg/day. Identifying prospectively the clinically-active dose ranges of erlotinib and gefitinib will help further personalize care for patients with tumors harboring EGFR mutations.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1784.
Abstract 1789: NFKBIA deletion in malignant gliomas
Authors: Bredel Markus , Yadav Ajay , Renfrow Jaclyn , Scholtens Denise , Chandler James , Bredel Claudia , Tagge Michael , Lukac Paul , Robe Pierre , Vogel Hannes , Scheck Adrienne , Aldape Kenneth , Chakravarti Arnab , Harsh Griffith
Published: February 2011

Introduction:

Aberrant activation of the Epidermal Growth Factor Receptor (EGFR) oncoprotein is a molecular hallmark of glioblastomas. Two-thirds of tumors with excessive EGFR activation show alteration of the EGFR gene. The mechanism of deregulation of EGFR in the other third remains poorly understood. Nuclear factor of κB inhibitor alpha (NFKBIA) is a potential tumor suppressor.

Methods:

Multidimensional molecular profiles for NFKBIA and EGFR and clinical profiles of 732 malignant glioma patients from multiple academic institutions, The Cancer Genome Atlas Pilot Project, the Repository of Molecular Brain Neoplasia Data, and a randomized phase III trial of temozolomide. NFKBIA mutational and functional analyses in a glioblastoma cell model.

Results:

We found that about 25% of glioblastomas harbor heterozygous NFKBIA gene deletions at chromosome 14q13. NFKBIA functions as a haploinsufficient tumor suppressor in glioblastomas by terminating EGFR signaling. Tumors with NFKBIA deletion lacked EGFR amplifications, indicating functional redundancy of both genetic events in glioblastoma pathogenesis. Loss of NFKBIA gene dosage was significantly associated with briefer patient survival. Patients with low NFKBIA expression had a significantly worse outcome than patients with intact NFKBIA expression. NFKBIA expression was also significantly associated with patient survival in subsets of tumors with unmethylated promoter of the O6-methylguanine DNA methyltransferase (MGMT) gene. A combined outcome predictor model of NFKBIA and MGMT proved highly predictive of duration of survival.

Conclusion:

Deletion of NFKBIA is an alternative mechanism for tumorigenic increase in EGFR signaling in glioblastomas. NFKBIA inactivation is associated with shorter survival of patients with malignant gliomas.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1789.
Abstract 179: Identification of novel targets for aberrant methylation in pancreatic cancer by a newly developed method “methyl-CpG targeted transcriptional activation (MeTA)”
Authors: Fukushige Shinichi , Shimizu Hideyuki , Sato Yuko , Horii Akira
Published: February 2011

Identification and characterization of genes selectively hypermethylated in cancer may improve our understanding of the role of epigenetic alterations in tumorigenesis. In addition, genes frequently methylated in a tumor-specific manner could be used as tumor markers. DNA methylation inhibitors such as 5-aza-cytidine or 5-aza-2’-deoxycytidine have been widely used to search epigenetically silenced genes. However, these inhibitors frequently upregulate genes whose promoters remain unmethylated. We tried to improve the specificity and sensitivity in detecting methylation-mediated silenced genes in cancer and successfully developed a new method termed “methyl-CpG targeted transcriptional activation (MeTA)” by using a transcriptional activating fragment with a methyl-CpG binding domain (MBD). Because MBD proteins in fact mediate transcriptional repression of tumor suppressor genes associated with promoter hypermethylation in cancer, MeTA is thought to be one of the ideal methods to search such genes. In the preliminary experiments using human embryonic kidney cell line 293T, we found that the combination of MeTA with gene expression microarray is useful; genes containing CpG islands can be efficiently identified (93.5% by MeTA vs. 66.8% by 5-aza-cytidine) even those that are difficult to find by the conventional DNA methylation inhibitors. We applied this method to twelve typical pancreatic cancer cell lines with a normal pancreatic ductal epithelial cell line HPDE as the control, and have searched for genes commonly upregulated after MeTA. Thirty-one genes were upregulated by two-folds or more in six pancreatic cancer cell lines or more. Among these, 26 genes have not been reported previously using the conventional DNA methylation inhibitors. Methylation statuses of three genes, NEFL, NEFM, and IRX4 were further examined by methylation-specific PCR and found that these three genes were aberrantly methylated in all of the analyzed tumors, 11 resected primary pancreatic cancer specimens and 22 pancreatic cancer cell lines. Our results suggest that “MeTA” is a highly efficient method to identify methylation-mediated transcriptionally silenced genes in human pancreatic cancer and that this method can be applied to other types of human cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 179.
Abstract 1794: TXNL2 is a key regulator of reactive oxygen species levels and NF-κB activity in cancer cells
Authors: Qu Ying , Wang Jinhua , Shin-Sim Miyung , Huang Jiang , Liu Bingya , Zhang Ning , Cui Yukung , Giuliano Armando E. , Cheng Ninghui , Cui Xiaojiang
Published: February 2011

Cancer cells usually generate and maintain higher levels of reactive oxygen species (ROS) compared to normal cells, in part because of their higher metabolic rate and defects in the electron transport chain. Because most tumor cells possess stronger antioxidative defense mechanisms to counterbalance excessive ROS, redox proteins such as thioredoxins (TRXs) and glutaredoxins (GRXs) are potential targets of anticancer therapy. We recently found that a novel redox protein called thioredoxin-like 2 (TXNL2) is required for embryonic stem cell functions and is consistently overexpressed in many different cancers. It has a TRX-homology domain and two GRX-homology domains, suggesting that it may have redox function.

Using immunohistochemical and cDNA microarray analyses, we found that levels of TXNL2 were significantly higher (P = 0.00036) in human breast cancers than in normal breast tissues; levels also were higher in human breast cancer cell lines than in normal mammary epithelial cell lines. Knockdown of TXNL2 by shRNA in breast cancer cell lines induced a 5-fold increase of ROS levels and inhibited cell proliferation, survival, invasion, and the expression of genes critically involved in these cellular processes. Breast cancer stem-like cell populations and stem-like properties such as mammosphere formation were reduced by TXNL2 silencing and induced by TXNL2 overexpression. Surprisingly, TXNL2 depletion in normal mammary epithelial cells did not significantly affect ROS levels and cell survival. Xenograft models of orthotopic injection indicated that TXNL2 knockdown inhibited mammary tumor growth and metastasis to the lung. Cell signaling studies showed that TXNL2 knockdown inhibited GSH synthase (GSS) expression, and thereby decreased GSH levels and increased ROS levels; N-acetylcysteine (NAC), a GSH precursor, partially reversed these effects. In addition, TXNL2 knockdown suppressed NF-κB activity by inactivating IKK and p65 via glutathionylation, whereas TXNL2 overexpression markedly increased NF-κB activity. Blockade of NF-κB using its small-molecule inhibitors and IκB overexpression mimicked the effect of TXNL2 depletion.

Our results suggest that TXNL2 is a potentially promising target for ROS-based anticancer therapies and is a key regulator of the NF-κB pathway. Because high cellular antioxidant capacity is well-known to impair cellular responses to chemotherapy and radiation therapy, our study may also open up a new avenue of maximize the efficacy of conventional anticancer therapies by targeting TXNL2.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1794.
Abstract 1795: The Combination of Paclitaxel and Gefitinib Inhibits Endometrial Cancer Cells by Inducing Mitotic Catastrophe: Proof of Principle for Dual Therapy in Endometrial Cancer
Authors: Meng Xiangbing , Laidler Laura L. , Albitar Lina , Holmes Anna M. , Dai Donghai , Buekers Thomas E. , Bender David P. , Leslie Kimberly K.
Published: February 2011

Serous uterine endometrial cancer is a lethal disease for which new therapeutic regimens are urgently needed. Combinations of chemotherapeutic agents and small molecule growth factor inhibitors have demonstrated activity in cancers from other sites. Our objective was to determine whether such a combination could be active in serous endometrial cancer cells. The effects of the EGFR inhibitor gefitinib (ZD1839, Iressa) alone, paclitaxel alone, and the combination of both agents on the cell cycle and on cell proliferation were studied using Hec50co cells, a validated model for type II serous endometrial cancer with absent p53. First, we established the IC50 for paclitaxel alone (14 nM) compared to that of paclitaxel and gefitinib in combination (1.3 nM). This 10-fold reduction in the IC50 with dual therapy yielded a combination index of 0.25, strongly suggesting that the paclitaxel and gefitinib combination resulted in synergistic growth inhibition. Sixty-seven percent of the cells treated with paclitaxel and gefitinib arrested in the G2/M phase. This arrest of the cell cycle at G2/M with combination therapy was significantly greater than with single agent treatment alone (p < 0.001) and was associated with inhibition of p38 phosphorylation and the induction of CDC25C phosphatase and Cyclin B1. When induced, these factors abrogate the G2/M checkpoint and result in rapid, premature progression to M phase; this mechanism is particularly active in the setting of absent p53. With rapid progression into M, the cells were highly sensitive to paclitaxel and were killed by the mechanism of mitotic catastrophe. Similar findings were observed in the same cells treated with the combination of paclitaxel and a specific inhibitor of p38, SB203580. Our study suggests that inhibition of EGFR or downstream p38 pathway can abrogate the mitotic stress checkpoint induced by cytotoxic agents targeting microtubules such as paclitaxel. These findings suggest a new therapeutic strategy for the treatment of serous endometrial carcinoma worthy of clinical confirmation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1795.
Abstract 180: Potential utility of Reprimo methylation as a sensitizer to anticancer treatment in gastric cancer
Authors: Akira Ooki , Yamashita Keishi , Watanabe Masahiko
Published: February 2011

Introduction: Reprimo is a highly glycosylated protein, inducing cell cycle G2 arrest. Reprimo promoter methylation has been reported as the mechanism regulating its expression in gastric cancer, but the biological roles remain unclear. The aim of this study is to examine Reprimo from a functional point of view.

Methods: The methylation status of Reprimo promoter was analyzed using quantitative-methylation specific polymerase chain reaction (Q-MSP), and the effects of expression were examined in gastric cancer cell lines (AZ521 and SH10) with no endogenous expression of Reprimo using stable transfection and phenotype analysis (WST assay and apoptosis assay).

Results: Reprimo methylation was detected in 6 of 8 gastric cancer cell lines; the silenced or reduced expression could be restored by treatment with demethylating agents such as 5-aza-2’-deoxycitidine and/or trichostatin A. On Q-MSP, the optimal cutoff value (0.42) determined by the receiver operating characteristic (ROC) curve could significantly discriminate primary tumor (69%, 57/83) from the corresponding normal tissues (18%, 15/83, P<0.0001). AZ521 cells with enforced expression significantly enhanced sensitivity to 5-FU therapy, and augmented apoptosis without affecting cell cycle. On the other hand, SH10 cells without methylation exhibited the undetectable level of endogenous expression, and Reprimo expression was robustly induced under 5-fluorouracil (5-FU), one of the standard anticancer agents for gastric cancer. As a result, SH10 cells stably transfected with Reprimo exhibited similar level apoptosis with mock cells.

Conclusion: Reprimo expression is inducible under 5-FU agent, that may lead to apoptosis in gastric cancer. However, its expression is frequently suppressed by promoter methylation. Thus, Reprimo methylation status is considered to have a great potential as a predictive biomarker to select patients who may be effective for 5-FU therapy in gastric cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 180.