bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3376: Mirk/Dyrk1B modulates the development of chemoresistance in human ovarian cancer.
Authors: Gao Jingchun , Li Ji , Gao Heli , Tian Jianxin , Lv Yunyi , Yang Zhihai , Zhang Qiuping , Kong Fandou , Pang Jian , Guan Hongwei , Shi Hong
Published: August 2014

Ovarian cancer is one of the leading causes of cancer death in women and chemoresistance is a major obstacle that need to be overcome in the treatment of ovarian cancer patients. Our previous study along with others have found that the serine/threonine kinase Mirk/Dyrk1B is overexpressed in a wide spectrum of cell lines and clinical specimens of ovarian cancer. It has been known that Mirk/Dyrk1B regulates cell survival and sensitivity to conventional chemotherapeutics, and it also contributes to G0 arrest by destabilization of cyclin D1 and stabilization of p27kip1in ovarian cancer cells. We therefore hypothesize that Mirk/Dyrk1B may be associated with cancer ‘stem cell-like’ sustaining cells and chemoresistance formation. In this study, the mRNA and protein expressions of Mirk/Dyrk1B and the side population (SP) cells in the human ovarian cancer SKOV3, OVCAR5, and A2780S cells were measured during cisplatin resistance development. In addition, the protein expressions of ALDH1, Nanog, Ki-67, and Mirk/Dyrk1B were examined in the clinical specimens obtained from the patients with high-grade serous ovarian cancer before and after neoadjuvant chemotherapy, respectively. It was found that drug resistance formation was accompanied by a significant increase in the mRNA and protein expressions of Mirk/Dyrk1B as well as proportion of SP cells in all ovarian cancer cell strains, which was annulled after knockdown of Mirk/Dyrk1B by shRNA. Moreover, the protein expressions of ALDH1, Nanog, and Mirk/Dyrk1B were up-regulated and ki-67 was down-regulated in the specimens after neodajuvant chemotherapy. Our result confirms the important contribution of Mirk/Dyrk1B-dependent mechanisms into the development of chemoresistance of human ovarian cancer.

Citation Format: Jingchun Gao, Ji Li, Heli Gao, Jianxin Tian, Yunyi Lv, Zhihai Yang, Qiuping Zhang, Fandou Kong, Jian Pang, Hongwei Guan, Hong Shi. Mirk/Dyrk1B modulates the development of chemoresistance in human ovarian 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 3376. doi:10.1158/1538-7445.AM2013-3376

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 338: Regulation of mutant p53 retarded cell growth established from mouse lung cancer.
Authors: Suh Young-Ah , Jung Chae Lim , Park Ji-Ho , Jang Se Jin
Published: August 2014

The genetic defects in lung cancer patient are the most important factor for malignancies, and it has been reported that approximately 50% of non-small cell lung cancer (NSCLC) patients have mutations in certain tumor-related genes. Therefore, exhaustive efforts have been devoted to uncovering the genetic alterations of lung cancer, although its driving genes and genetic determinants that attribute to the development of lung cancer so far remain elusive.

The K-ras activation due to the mutant is notorious in many kinds of cancer types, including pancreatic cancer and lung adenocarcinoma, as is generally accepted as driving mutation for lung malignancy of lung adenocarcinoma. Another predominantly alternated gene in lung cancer is the EGFR that hits the similar signaling with K-ras activation, to which small molecules and antibodies target to halt the tumor progression. The tumor suppressor p53 is also often altered in almost all kinds of cancers and lung cancer. In this reason, K-ras activated mouse is utilized as lung cancer mouse model. K-ras activated mouse eventually develops lung adenocarcinoma, which can be accelerated with mutated p53.

Due to the gain-of-function, mutant p53 protein contributes to tumor metastasis and shortening of the latency of tumor progression. Thereby activated K-ras/mutant p53 mouse is preferentially used as lung adenocarcinoma preclinical model. Here, we have regulated mutant p53 protein expression in mouse embryo fibroblast (MEF) or tumor cells derived from activated K-ras/mutant p53 mouse with small molecules to inhibit tumor growth. The cell growth was inhibited when the mutant p53 was destabilized. We assessed the mechanism that is responsible for the regulation of gain-of-function metastatic phenotype observed in the p53 mutant mouse

Citation Format: Young-Ah Suh, Chae Lim Jung, Ji-Ho Park, Se Jin Jang. Regulation of mutant p53 retarded cell growth established from mouse 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 338. doi:10.1158/1538-7445.AM2013-338
Abstract 3380: Overcoming platinum resistance in ovarian cancer using the novel compound MLN4924.
Authors: Jazaeri Amir A. , Shibata Etsuko , Park Jonghoon , Bryant Jennifer , Conaway Mark R. , Modesitt Susan C. , Smith Peter G. , Milhollen Michael , Berger Allison J. , Dutta Anindya
Published: August 2014

Purpose: The objective of this investigation was to evaluate the preclinical activity of MLN4924, an investigational inhibitor of the NEDD8-activating enzyme, against chemoresistant and chemosensitive ovarian cancer cells.

ExperimentalDesign: Efficacy of MLN4924 both alone and in combination with cisplatin was assessed using short-term tetrazolium based and longer term focus formation assays. Mechanistic studies included cell cycle analysis, evaluation of DNA-platinum adduct formation (using an ELISA), and evaluation of DNA damage repair pathways. In addition, we employed an siRNA screen to assess the contribution of each member of the Cullin RING-Ligase (CRL) family of E3 ubiquitin ligases, the best characterized downstream mediators of MLN4924’s biological effects.

Results: Single agent MLN4924 exhibited moderate activity in ovarian cancer cell lines. The combination of MLN4924 with cisplatin or carboplatin resulted in significant platinum sensitization in SKOV3 and ES2 cells, as well as, in several primary ovarian cancer cell lines established from high grade serous, clear cell, and serous borderline ovarian tumors. Furthermore, the platinum sensitizing effect of MLN4924 was also observed in several in vitro models of platinum resistant ovarian cancer (using both established and primary chemoresistant ovarian cancer cells). Mechanistically, we show that depletion of CUL3 and CUL5 augment, while knockdown of CUL1 and CUL4A antagonize cisplatin-induced cytotoxicity in ovarian cancer cells. The combination of cisplatin and MLN4924 was not associated with DNA re-replication, altered platinum-DNA adduct formation, or abrogation of FANCD2 monoubiquitination.

Conclusions: Our investigation reveals a novel mechanism for overcoming platinum resistance in vitro, and provides a strong rationale for clinical investigations of platinum and MLN4924 combinations in ovarian cancer. This work also highlights that different CRL ubiquitination pathways can have distinct and opposing effects on platinum sensitivity and resistance in ovarian cancer cells.

Citation Format: Amir A. Jazaeri, Etsuko Shibata, Jonghoon Park, Jennifer Bryant, Mark R. Conaway, Susan C. Modesitt, Peter G. Smith, Michael Milhollen, Allison J. Berger, Anindya Dutta. Overcoming platinum resistance in ovarian cancer using the novel compound MLN4924. [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 3380. doi:10.1158/1538-7445.AM2013-3380
Abstract 3389: Nuclear accumulation of c-Rel transcription factor is correlated with the proteasome inhibitor resistance in mantle cell lymphoma.
Authors: Blonska Marzenna , Pan Deng , Ou Zhishuo , Zhang Liang , Lin Xin , Wang Michael
Published: August 2014

Purpose: Nuclear factor kB (NF-kB) plays essential roles in survival and drug resistance of multiple malignancies including mantle cell lymphoma (MCL) and multiple myeloma (MM). Proteasome inhibitors have been shown to block constitutively active NF-kB and clinical data indicates a high overall response of MCL and MM patients to this treatment. The 2nd generation proteasome inhibitor, carfilzomib (CFZ), has a therapeutic effect on relapsed or refractory disease and low toxicity to normal lymphocytes. However, there are increasing numbers of proteasome inhibitor-resistant cases. Thus, this study was designed to identify the molecular mechanism that leads to CFZ-resistance.

Experimental design: In this study we have determined the cytotoxic effect of CFZ in a panel of MM and MCL cell lines, as well as primary tumor cells. The cell growth inhibition was assessed by the thymidine intake and MTT assay. NF-kB activity was examined by electrophoretic mobility shift assay (EMSA) using nuclear extracts from CFZ-treated and control cells. Nuclear pattern of NF-kB family members, such as RelA/p50, cRel/p50 and RelB/p52 complexes, was analyzed by supershift and western blot.

Results: CFZ-induced NF-kB inhibition was not observed in two (out of eight) examined cell lines, even with the highest doses (100 nM). Consistently, cell growth was suppressed only in cells with a CFZ-sensitive NF-kB activity (IC50<20 nM), as shown by EMSA and MTT assay. In contrast, the treatment with proteasome inhibitor increased the nuclear accumulation and DNA binding of NF-kB in CFZ-resistant cells. Furthermore, we have found that only these cells had upregulated c-Rel/p50 member of NF-kB family. Supershift analysis revealed that all examined cell lines and primary cells had constitutively active classical (RelA/p50 dimer) and/or non-classical (RelB/p52 dimer) NF-kB, excluding CFZ-resistant cells with prominent nuclear accumulation of c-Rel. This data suggests that activated c-Rel may trigger resistance to CFZ. Enhanced nuclear accumulation of c-Rel is also an unwanted molecular consequence of exposure to CFZ.

Conclusion: Our work indicates the need for new treatment strategies in c-Rel-positive cases. A strategic combination targeting different signaling mediators, especially upstream of c-Rel, would enhance the ultimate anticancer effect.

Citation Format: Marzenna Blonska, Deng Pan, Zhishuo Ou, Liang Zhang, Xin Lin, Michael Wang. Nuclear accumulation of c-Rel transcription factor is correlated with the proteasome inhibitor resistance in mantle cell lymphoma. [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 3389. doi:10.1158/1538-7445.AM2013-3389
Abstract 3391: Overexpression of Nek2 promotes bortezomib resistance in multiple myeloma cells.
Authors: Flory Jessica , Brog Rachel , Meng Ling Yao , Call Lee , Vankayalapati Hariprasad , Tricot Guido , Zhan Fenghuang , Bearss David J.
Published: August 2014

Drug resistance is a major complication in cancer therapy but the molecular mechanisms driving both innate and acquired drug resistance still remain largely unknown for many anti-cancer agents. Microarray analyses on paired primary multiple myeloma samples at baseline and after therapy or at relapse showed that the mitotic kinase NEK2 was one of the most up-regulated genes in myeloma cells after high-dose chemotherapy or at relapse. Nek2 is a Serine/Threonine kinase that is activated during mitosis and is associated centrosome function and structure at the G2/M transition. A role for Nek2 has been suggested in the separation of centrosomes as well as promoting cell cycle from G2 phase to M phase but no role for Nek2 in mediating drug resistance has been described. By analyzing the published (> 2,500) microarrays and clinical datasets, we found that NEK2 expression is increased in many malignancies, and that high expression of NEK2 was associated with a shorter event-free and overall survival. Moreover, NEK2 expression was typically increased in tumors with aggressive subtype and advanced stage. Our studies in myeloma cells indicate that over-expressing NEK2 results in enhanced cell proliferation and drug resistance, whereas knockdown of NEK2 induced significant cancer cell death and growth inhibition. Nek2 transfected myeloma cell lines ARP1, H929 and KMS28PE were used to investigate Nek2’s function relative to bortezomib resistance. We found that Nek2 over-expression significantly elevated the proteasome activity of transfected cells leading to decreased activity of Bortezomib in these cells. Using novel small molecule Nek2 inhibitors developed in our lab, we demonstrated efficient inhibition of proteasome activity in Nek2 over-expressing cancer cell lines. The treatment of cell with our lead compounds significantly inhibited the degradation of some proteasome targeted cell cycle regulators. Further, the combinations of these Nek2 inhibitors and bortezomib, greatly elevated the efficiency of bortezomib in decreasing the proteasome activity in vitro. The treatment with these novel Nek2 inhibitors successfully rescued the drug resistance in Nek2 over-expressed cells, indicating a potential therapy for bortezomib resistant myeloma patients. We conclude that NEK2 represents a predictor for drug resistance and poor prognosis in cancers and could be a potential target for cancer therapy.

Citation Format: Jessica Flory, Rachel Brog, Ling Yao Meng, Lee Call, Hariprasad Vankayalapati, Guido Tricot, Fenghuang Zhan, David J. Bearss. Overexpression of Nek2 promotes bortezomib resistance in multiple myeloma 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 3391. doi:10.1158/1538-7445.AM2013-3391
Abstract 3392: New findings of kinase switch in gastrointestinal stromal tumor under imatinib using phosphoproteomic analysis.
Authors: Ako Maiko , Takahashi Tsuyoshi , Serada Satoshi , Fujimoto Minoru , Miyazaki Yasuaki , Shimada Kazuki , Ikezoe Takayuki , Taguchi Takahiro , Kurokawa Yukinori , Yamasaki Makoto , Miyata Hiroshi , Nakajima Kiyokazu , Takiguchi Shuji , Mori Masaki , Doki Yuichirou , Naka Tetsuji , Nishida Toshirou
Published: August 2014

Despite the initial effectiveness of imatinib for gastrointestinal stromal tumors (GISTs), patients must continue lifelong imatinib therapy, and may eventually relapse owing to acquired drug resistance. Pathology reveals the persistence of viable cells during imatinib therapy, even in the disappearance region upon radiological examination. These cells are thought to cause acquired drug resistance and relapse. To uncover the mechanisms underlying these clinical issues, we used GIST-T1 cells to investigate the phosphoproteomic alterations induced by imatinib treatment. Because some tyrosine kinases might be activated after imatinib exposure, it is important to reveal the increased phosphorylation of proteins on a proteomic scale, and elucidate drug-resistant proteins. Proteins extracted from imatinib-treated or untreated GIST-T1 cells were digested with trypsin. The resulting tyrosine-phosphorylated peptides were purified using an immunoaffinity-enriched and iTRAQ-labeled reagent, and analyzed using mass spectroscopy. Using this phosphoproteomic approach, we identified 171 tyrosine phosphorylation sites spanning 134 proteins. We also detected increased tyrosine phosphorylation of Fyn (Y420) and focal adhesion kinase (FAK) (Y576). Increased phosphorylation was also confirmed by Western blot analysis, and knockdown of Fyn or FAK increased cells’ sensitivity to imatinib. Furthermore, the anti-proliferative effect of imatinib significantly enhanced imatinib-resistant GIST-T1 cells, which exhibited constitutive phosphorylation of FAK when treated with a FAK-selective kinase inhibitor. Moreover, FYN was proved to be a signal of FAK of upper reaches.

These results indicate that an iTRAQ-based quantitative phospho-tyrosine-focused phosphoproteomic approach is a powerful method for screening phosphoproteins associated with drug resistance. Fyn or FAK might be potential therapeutic targets to overcome acquired resistance to imatinib in GISTs.

Citation Format: Maiko Ako, Tsuyoshi Takahashi, Satoshi Serada, Minoru Fujimoto, Yasuaki Miyazaki, Kazuki Shimada, Takayuki Ikezoe, Takahiro Taguchi, Yukinori Kurokawa, Makoto Yamasaki, Hiroshi Miyata, Kiyokazu Nakajima, Shuji Takiguchi, Masaki Mori, Yuichirou Doki, Tetsuji Naka, Toshirou Nishida. New findings of kinase switch in gastrointestinal stromal tumor under imatinib using phosphoproteomic analysis. [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 3392. doi:10.1158/1538-7445.AM2013-3392
Abstract 3397: Synergistic activity of the Akt inhibitor perifosine and 5-fluorouracil in 5-FU resistant gastric cancer.
Authors: Hyo Song Kim , Shin You Keun , Zhang Xianglan , Kim Jung Min Kim , Kim Tae Soo , Park Chan Hee , Jang Woo Ick , Engel Juergen , Rha Sun Young Rha
Published: August 2014

Purpose

Akt/PI3K pathway has been known to confer resistance to conventional chemotherapy. Perifosine is a synthetic novel alkylophospholid which inhibits Akt activation. Because 5-fluorouracil (5-FU) is a common cytotoxic agent used in gastric cancer, we evaluated the role of perifosine overcoming 5-FU chemoresistance.

Experimental design

We checked the correlation between pAkt expression and 5-FU sensitivity in 25 gastric cancer cell lines. Cytotoxic effects of 5-FU and perifosine combination were assessed using in vitro assays (cell growth and apoptosis analysis). Whether this synergistic combination of two drugs has significant effect on their downstram targets and on biochemical markers were also tested with western blotting analysis. Tumor inhibition and pAkt expression were measured using xenograft mice treated with perifosine and 5-FU combination.

Results

More 5-FU resistant cell lines demonstrated higher pAkt expression levels (mean 1.35 IU vs 0.49 IU, p=0.019). Among the 14 5-FU resistant cell lines, majority of the cell lines (57.1%) showed synergistic effect with perifosine combination. Perifosine and 5-FU combination caused marked Akt and extracellular signal-regulated kinase (Erk) 1/2 dephosphorylation in synergistic cell line. Moreover, combination results in a substantial induction of apoptosis by JNK pathway activation in synergistic cell line. In vivo model, treatment with tolerated doses of 5-FU derivatives, capecitabine and perifosine resulted in significantly enhanced antitumor efficacy relative to that of either agent alone (P=0.006). Histopathologic analysis of pAkt expression also revealed prominent pAkt reduction in combination groups. In the gene expression profiling analysis, Akt1 and MAPK4K genes were up-regulated in synergistic cell lines implicating those genes as predictive markers for restoring 5-FU resistance in combination treatment.

Conclusions

These results indicate that activated Akt pathway confers 5-FU chemoresistance. In addition, perifosine and 5-FU combination restored those chemoresistance via pAkt, pErk dephosphorylation and JNK activation. Taken together, 5-FU and perifosine combination may be a promising therapeutic strategy to improve patient outcome in gastric cancer patients.

Citation Format: Kim Hyo Song, You Keun Shin, Xianglan Zhang, Jung Min Kim Kim, Tae Soo Kim, Chan Hee Park, Woo Ick Jang, Juergen Engel, Sun Young Rha Rha. Synergistic activity of the Akt inhibitor perifosine and 5-fluorouracil in 5-FU resistant gastric 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 3397. doi:10.1158/1538-7445.AM2013-3397

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 3403: A stochastically arising subpopulation of B-RafV600E-expressing melanoma continues division in the presence of vemurafenib.
Authors: Tyson Darren R. , Peng Chengwei , Hardeman Keisha N. , Paudel Bishal , Quaranta Vito
Published: August 2014

Patients with melanomas expressing B-RafV600E benefit from treatment with B-Raf-targeted therapeutics, such as vemurafenib. Initial responses to these agents are occasionally profound, but the duration of response is variable from patient to patient, with some tumors recurring within a few weeks and others requiring months before tumors progress. The source of this variability of response is under intense investigation. A number of acquired resistance mechanisms have been described, but it is not known whether they pre-exist or develop after treatment initiation. To address this question, we tracked the cellular response to PLX4720 (the vemurafenib research compound) over 96 hours in the A375 and SK-MEL-5 cell line models of B-RafV600E-expressing melanoma. Both cell lines exhibited a significant early reduction in cell number compared to control, but after 60 hours of treatment cells numbers began to increase even in the presence of 8 μM drug, albeit at a slow rate. This “rebound” proliferation effect was not due to drug decay since re-administration of the drug did not prevent it. We further examined the rebound effect at the single cell level using our newly described Fractional Proliferation assay (Tyson et al., Nat Methods, 2012, 9(9):923-928, doi:10.1038/nmeth.2138). The rebound corresponded to a dividing cell subpopulation whose size and rate of division depend on the concentration of drug. This subpopulation does not represent rare non-responding variants within the cell lines, but rather it arises stochastically as sibling cells do not always exhibit the same phenotypic response. Investigation of the molecular correlates of this dividing subpopulation, which may act as the reservoir from which the resistant cells arise, are ongoing.

Citation Format: Darren R. Tyson, Chengwei Peng, Keisha N. Hardeman, Bishal Paudel, Vito Quaranta. A stochastically arising subpopulation of B-RafV600E-expressing melanoma continues division in the presence of vemurafenib. [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 3403. doi:10.1158/1538-7445.AM2013-3403
Abstract 3406: Evaluation of (E)-Styrylsulfonyl methylpyridine: A novel kinase inhibitor targeting mitotic pathways.
Authors: Lu Tiangong , Wang Shudong , Laughton Charles , Bradshaw Tracey
Published: August 2014

ON01910.Na, a styryl benzylsulfone, is a Phase III stage, non-ATP competitive anti-cancer agent. It is multi-targeted, promoting selective mitotic arrest and apoptosis in cancer cells. Extensive Phase II/III trials conducted in patients with solid tumors and hematological cancers have proved its excellent efficacy and impressive safety profile. However, incomplete understanding of mechanisms of action and relatively low drug oral bioavailability remain obstacles to development. By modifying the structure of ON01910.Na, a novel class of (E)-Styrylsulfonyl methylpyridines was designed and synthesized. We report herein the evaluation of a selected compound, TL-77, (E)-N-(2-methoxy-5-((2,4,6-trimethoxystyrylsulfonyl)methyl)pyridin-3-yl)methanesulfonamide, as a potent small molecule inhibitor targeting mitotic pathways.

MTT assays were performed to assess in vitro anti-proliferative effects. In a panel of 10 tumor and 2 non-transformed human cell lines following 96 h exposure, TL-77 gave consistent GI50 values in the sub-micromolar range in tumor cells, whereas, it was non-toxic to non-transformed human mammary endothelial cells (HMECs). Cell cycle was analyzed by flow cytometry. TL-77 caused significant G2/M arrest ≥ 6 h, whereas ON01910.Na caused the same effect after longer exposure periods (≥ 12 h). An OD-based tubulin polymerization assay allowed us to explain obstruction of cell division caused by TL-77. TL-77 retarded tubulin polymerization between 1μM and 6μM, intriguingly, microtubules were stabilizes at TL-77 concentrations > 8μM. Western blot analyses of cell lysates following treatment of cells with TL-77 revealed dose-dependent reduction of phosphorylated Cdc25c, a known substrate of Plk1 and/or Chk1/2, indicating Plk1 and/or Chk1/2 inhibition. Finally, dose- and time-dependent apoptosis triggered by TL-77, detected by Annexin-V assays, was closely associated with induction of caspase 3/7 activity. Importantly, pharmacokinetics studies in mice revealed that the oral bioavailability of TL-77 was optimized to 56% compared with 9% for ON01910.Na.

In summary, TL-77 is a potent anti-cancer agent. It disturbs tubulin during cell division, resulting in G2/M cell cycle arrest, followed by caspase-dependent apoptosis. TL-77 appears to share an analogous mechanism of action with ON01910.Na, but exhibits greater selectivity towards cancer cells and possesses superior oral bioavailability. Therefore, further evaluation of TL-77 as a promising anti-tumor agent is justified.

Citation Format: Tiangong Lu, Shudong Wang, Charles Laughton, Tracey Bradshaw. Evaluation of (E)-Styrylsulfonyl methylpyridine: A novel kinase inhibitor targeting mitotic pathways. [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 3406. doi:10.1158/1538-7445.AM2013-3406
Abstract 341: In vivo model of non-small cell lung cancer metastasis to the brain.
Authors: Chung Su I. , Tobin Lisa A. , Xie Yili , Wunderlich John R. , Kwong King F.
Published: August 2014

Background: Non-small cell lung cancer (NSCLC) has a high rate of metastasis to distant sites such as the brain. Previous reports have suggested that exogenously expressed EMT transcription factors lead to EMT-induced migration, invasion, and anchorage-independent growth of cancer cells in vitro. In other cancer types, increased EMT transcription factors leads to promotion of metastatic tumor cell dissemination but simultaneously can also inhibit distant colonization by circulating metastatic cells. Methods: To investigate the role of EMT in brain metastasis in NSCLC, we established an in vivo model of brain metastasis through the isolation of brain metastatic clones from iterative rounds of systemic cancer cell inoculation in immunodeficient mice. Results: With each round, an increasing frequency of brain metastasis characterized the metastatic pattern and earlier onset of brain tumor formation was seen in select animals. In the metastatic cell lines established from the explanted brain metastases, we observed elevation of EMT markers such as TWIST, vimentin, and fibronectin compared to the parental cells. In addition, metastatic clones to the brain demonstrated markedly increased migration and EGFR expression. Conclusion: Iterative in vivo passaging of cancer cells facilitated isolation of subclones demonstrating increased brain metastasis. Also, these data suggests that the increased expression of EMT markers does not inhibit the formation of brain metastasis in NSCLC.

Citation Format: Su I. Chung, Lisa A. Tobin, Yili Xie, John R. Wunderlich, King F. Kwong. In vivo model of non-small cell lung cancer metastasis to the brain. [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 341. doi:10.1158/1538-7445.AM2013-341
Abstract 3410: Inhibitors of Skp2 E3ligase stabilize nuclear p27kip1 for regain of growth regulation in cancer.
Authors: Pavlides Savvas C. , Huang Kuang-Tzu , Wu Lily , Rueda Bo R. , Blank Stephanie V. , Mittal Khushbakhat R. , Cardozo Timothy , Gold Leslie I.
Published: August 2014

In many human cancers, the tumor suppressor, p27kip1 (p27), a cyclin-dependent kinase inhibitor critical to cell cycle arrest, undergoes perpetual ubiquitin-mediated proteasomal degradation by the E3 ligase complex SCF-Skp2/Cks1 and/or cytoplasmic mislocalization; lack of nuclear p27 causes aberrant cell cycle progression and cytoplasmic p27 can mediate cell migration/metastasis. We previously showed that estrogen (E2) induces Skp2-dependent degradation of p27 and cell proliferation in primary endometrial epithelial cells (EECs) and endometrial carcinoma (ECA) cell lines (e.g., ECC-1) suggesting a pathogenic mechanism for type I endometrial carcinoma (ECA), an estrogen (E2)-linked cancer. The current studies show that treatment of ECC-1 cells with small molecule inhibitors of Skp2/Cks1 E3 ligase activity (Skp2E3LIs) increase protein levels of p27, increase p27 half-life by 6 hours and inhibit cell proliferation (IC50 10μM). Two of five SKP2E3LIs, designated as C2 and C20 specifically increase p27 in the nucleus while decreasing p27 in the cytoplasm in both ECC-1 cells and primary ECA cells. In addition, C2 and C20 prevent both E2-induced proliferation and degradation of nuclear p27. These data suggest that Skp2E3LIs can function in the nucleus to prevent E2-induced degradation of p27 in primary ECA cells to regain growth control by directly blocking p27 ubiquitylation and subsequent degradation. Skp2E3LIs enable a chemical biology approach to understanding the functional significance of the ubiquitin pathway in p27-mediated cell cycle regulation and importantly, are the first specific proteasome inhibitors that pharmacologically target the binding interaction between the E3ligase, SCF-Skp2/Cks1 and p27 to prevent its ubiquitylation and subsequent degradation. These targeted inhibitors represent a major therapeutic advancement over general proteasome inhibitors for cancers characterized by SCF-Skp2/Cks1-mediated destruction of p27.

Citation Format: Savvas C. Pavlides, Kuang-Tzu Huang, Lily Wu, Bo R. Rueda, Stephanie V. Blank, Khushbakhat R. Mittal, Timothy Cardozo, Leslie I. Gold. Inhibitors of Skp2 E3ligase stabilize nuclear p27kip1 for regain of growth regulation in 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 3410. doi:10.1158/1538-7445.AM2013-3410
Abstract 3412: Imaging of methioninase-induced S/G2-phase-trapping for subsequent effective chemotherapy.
Authors: Yano Shuya , Tome Yasunori , Digman Michelle , Momiyama Masashi , Suetsugu Atsushi , Gratton Enrico , Hoffman Robert M.
Published: August 2014

Methionine-dependence of cancer cells may be due to excessive methylation reactions in cancer cells. Deprivation of methionine α,γ lyase (methioninase or METase) selectively arrests cancer cells during late S-phase, where they are highly sensitive to DNA-damaging chemotherapy. Fluorescent ubiquitination-based cell cycle indicator (FUCCI)), was used to monitor the onset of the S/G2-phase block due to methionine deprivation effected by METase. The S-phase-blocked cancer cells fluoresced yellow or green, in contrast to cancer cells in G1 which fluoresced red. Cancer cells, synchronously blocked in S-phase by METase and identified by their yellow-green fluorescence, were administered DNA-damaging chemotherapy drugs such as doxorubicin, cisplatin, or 5-fluorouracil. Treatment of cancer cells with drugs only without methioninase-effected S-phase synchrony, led to the majority of the cancer cell population being blocked in G0/G1 phase (red fluorescent) where they were resistant to the drugs. In contrast, METase treatment, followed by chemotherapy when FUCCI indicated the S/G2 block was highly effective for killing cancer cells. Color-coded chemotherapy, whereby the cell cycle of cancer cells is selectively and synchronously blocked in S-phase as identified by fluorescent reporters, may be a general approach to effective cancer treatment.

Citation Format: Shuya Yano, Yasunori Tome, Michelle Digman, Masashi Momiyama, Atsushi Suetsugu, Enrico Gratton, Robert M. Hoffman. Imaging of methioninase-induced S/G2-phase-trapping for subsequent effective chemotherapy. [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 3412. doi:10.1158/1538-7445.AM2013-3412
Abstract 3414: Disruption of CREB-CREBBP association by 2-Naphthol AS-E phosphate induces cell cycle arrest and apoptosis in non-small cell lung cancer cells .
Authors: Lee Jong Woo , Park Hee Sun , Ryu Seung-Hee , Koch Patricia , Boyer Julie , Morgensztern Daniel , Herbst Roy S. , Koo Jaseok Peter
Published: August 2014

The cyclic-AMP response element-binding protein (CREB) plays a critical role in the growth and survival of cancer cells as well as cell metabolism. Our previous studies have shown that enhanced CREB activity correlates with increased growth and survival of non-small cell lung cancer (NSCLC) cell lines. In addition, 2-naphthol AS-E phosphate (NASEp), a small molecule inhibitor of CREB, restricted growth of NSCLC cell lines which was associated with reduced expression of pro-angiogenic factors. NASEp distrupts binding of CREB binding protein (CREBBP) to CREB, however, the mechanism which CREB inhibition by NASEp regulates NSCLC cell growth and survival is not fully understood. Here, we found that NASEp inhibited cell growth, survival, anchorage-independent cell growth and invasion, and also completely blocked the expression of Ki-67 and PCNA, markers of cell proliferation in NSCLC cells. Mechanistically, NASEp caused cell cycle arrest at the sub-G1 or G2 phase through down-regulation of several cyclins (cyclin A2, cyclin B1, cyclin D1 and cyclin E2) and led to apoptosis in NSCLC-derived cell lines. Suppression of cyclin D1 was closely related to decreased expression of activator protein-1 (AP-1). Furthermore, in a murine xenograft model, NASEp significantly reduced tumor burden not only by down-regulating of cyclin E2 and PCNA, but also by induction of apoptosis. Taken together, these results suggest that inhibition of CREB activity through dissociation of CREB-CREBBP by NASEp may be a potent new therapeutic in NSCLC.

Citation Format: Jong Woo Lee, Hee Sun Park, Seung-Hee Ryu, Patricia Koch, Julie Boyer, Daniel Morgensztern, Roy S. Herbst, Jaseok Peter Koo. Disruption of CREB-CREBBP association by 2-Naphthol AS-E phosphate induces cell cycle arrest and apoptosis in non-small cell lung 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 3414. doi:10.1158/1538-7445.AM2013-3414
Abstract 3425: Chk1 inhibitor targets replicative stress in melanomas.
Authors: Gabrielli Brian G. , Brooks Kelly , Edwards Brooke , Chen James , Pavey Sandra
Published: August 2014

There are few effective treatments for metastatic melanoma. Chk1 inhibitors are being trialed for their efficacy in enhancing conventional chemotherapeutic agents, but their effectiveness as single agents is relatively unexplored. We have examined the effectiveness of two novel Chk1 selective inhibitors, AR-323 and AR-678, in a large panel (40) of melanoma cell lines and normal cell types in vitro, and investigated the mechanism promoting their cytotoxicity in the drug-sensitive cell lines. We demonstrate that these drugs display single agent activity, with IC50s in the nanomolar range. The drugs produce cytotoxic effects in cell lines that are most sensitive to these drugs, whereas normal cells are only sensitive to these drugs at the higher concentrations where they have cytostatic activity. The cytotoxic effect is related to inhibition of S phase Chk1 which drives cells prematurely from late S phase, resulting in apoptosis either prior to or during an aberrant mitosis. Time lapse imaging has shown that the timing of cell death is related to the drug sensitivity of the cell line. The sensitivity to the Chk1 inhibitors was correlated with the level of endogenous DNA damage indicating replicative stress, and increasing endogenous replicative stress increased drug sensitivity. Chk1 inhibitors are viable single agent therapies which target melanoma cells with high levels of endogenous DNA damage. This sensitivity suggests that Chk1 is a critical component of an adaptation to replicative stress in these cells. It also suggests that markers of DNA damage may be useful in identifying the melanomas and potentially other tumour types that are more likely to be sensitive to Chk1 inhibitors as single agents.

Citation Format: Brian G. Gabrielli, Kelly Brooks, Brooke Edwards, James Chen, Sandra Pavey. Chk1 inhibitor targets replicative stress in melanomas. [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 3425. doi:10.1158/1538-7445.AM2013-3425
Abstract 3427: The dinuclear platinum agent, BBR3610-DACH, exhibits a significant anti-proliferative effect: Regulation of G1/S and G2/M cell cycle arrest and apoptosis induction.
Authors: Menon Vijay , Peterson Erica , Deb Sumitra , Povirk Lawrence , Farrell Nicholas
Published: August 2014

BBR3610-DACH, a long-chain, bifunctional dinuclear PTII complex with a similar DNA binding mode to the Phase II clinical drug, BBR3464 and its potent analogue, BBR3610, was shown previously to be resistant to metabolic decomposition by sulfur-containing nucleophiles. Initial observations utilizing the alkaline comet assay indicate that BBR3610-DACH forms interstrand crosslinks. Cell cycle analysis using flow cytometry showed that BBR3610-DACH inhibited cell proliferation and caused G1/S and G2/M cell cycle arrest in HCT116 colorectal cells in a time dependent manner accompanied with significant s phase depletion. Similar experiments with synchronized cells showed a distinct G1 phase arrest elicited by the drug following release from arrest. Immunoblotting revealed a stabilization of p53 between 6-24hr after treatment with 20μM (IC90) BBR3610-DACH, with a concomitant increased levels of the cyclin dependent kinase inhibitors, p21 and p27. Cell proliferation assay using MTT and subsequent cell cycle studies with HCT116 p53-/- and HCT116 p21-/- indicated that the cytotoxicity profile of BBR3610-DACH was p21 dependent and partially dependent on the p53 status. However, no change was observed in the protein levels of cyclin E, CDK2, and CDC25A which are G1 phase regulators. Decrease in the levels of cyclin A and cyclin B1 at 24hrs suggested a G2/M block indicative of a possible mitotic catastrophe. Next, we examined whether the DNA damage sensor protein kinases, ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and RAD3 related (ATR) had a role in the cell cycle regulation after drug treatment. Studies using the ATM inhibitor, KU-60019 and caffeine (inhibits both ATM and ATR) revealed no apparent role of ATM but a possible dependence on ATR. Following the cell cycle effects, BBR3610-DACH induced cell death as seen from cleaved poly (ADP-ribose) polymerase-1 (PARP-1) levels as early as 6hrs after drug treatment. Interestingly, no cleaved caspase 3 was seen suggesting a caspase-independent PARP cleavage. Together these findings suggest that BBR3610-DACH is a major cell cycle inhibitory DNA binding platinum agent that could be further developed as a major chemotherapeutic.

Citation Format: Vijay Menon, Erica Peterson, Sumitra Deb, Lawrence Povirk, Nicholas Farrell. The dinuclear platinum agent, BBR3610-DACH, exhibits a significant anti-proliferative effect: Regulation of G1/S and G2/M cell cycle arrest and apoptosis induction. [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 3427. doi:10.1158/1538-7445.AM2013-3427
Abstract 3428: Effect of a non cardiotoxic Doxorubicin analog, 13-deoxy, 5-imino doxorubicin on decatenation of DNA by Topoisomerase II.
Authors: Frank Nicole , Olson Richard , Walsh Gerald M. , Talley Todd , Cusack Barry
Published: August 2014

Anthracyclines such as doxorubicin are widely used to treat solid and hematological cancers. However, within the course of treatment they have the potential to cause life threatening chronic cardiotoxicity which limits their potential use. The cause of cardiotoxicity is not thoroughly understood, with previous research indicating the generation of reactive oxygen species and free iron accumulation as possible causes for cardiomyocyte death. Recent publications have shown the interaction between doxorubicin and Topoisomerase IIβ plays a key role in anthracycline induced cardiotoxicity. Topoisomerase enzymes are utilized during the coiling and uncoiling of DNA within the cell and are active targets of anthracyclines not only in tumoricidal action (Topoisomerase IIα) but also in the pathogenesis of cardiotoxicity (Topoisomerase IIβ). In this research we seek to further evaluate the interaction between doxorubicin, doxorubicin analogues and Topoisomerase IIβ. The analogues were selected due to previous chronic in vivo experiments in rabbits where classical manifestations of cardiotoxicity (including reduced left ventricular shortening fraction and pathological changes in cardiac tissue) were not observed. Furthermore, one analogue (13-deoxy, 5-imino doxorubicin) has shown promising clinical data suggesting a strong antineoplastic effect without the cardiotoxic effects. Doxorubicin and this analogue were placed in the presence of catenated DNA for 6 hours at 37°C prior to exposure to Topoisomerase IIα and β enzymes. The reaction was allowed to proceed for 30 minutes at 37°C and analyzed for decatenation of the DNA by the enzyme. This experiment was then repeated in cardiac H9c2 cells to assay possible differential inhibition at the cellular level.

Citation Format: Nicole Frank, Richard Olson, Gerald M. Walsh, Todd Talley, Barry Cusack. Effect of a non cardiotoxic Doxorubicin analog, 13-deoxy, 5-imino doxorubicin on decatenation of DNA by Topoisomerase II. [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 3428. doi:10.1158/1538-7445.AM2013-3428
Abstract 3429: Coordination of cell cycle repression and reduced cytoskeletal tension by the small molecule natural product, Schweinfurthin A.
Authors: Turbyville Thomas , Brafman Alla , Chen De , Romanchuck Kathryn , Beutler John , Reilly Karlyne , Lockett Stephen
Published: August 2014

Malignant Peripheral Nerve Sheath Tumors (MPNST) are a frequent cause of morbidity and mortality in Neurofibromatosis type 1 patients, and arise in part from loss of the tumor suppressor function of the NF1 gene product, neurofibromin. We have previously shown that MPNST tumor cell lines, as well as astrocytoma cell lines, derived from both patients and a Nf1-/+;Trp53-/+ (NPcis) mouse model are differentially sensitive to the natural product, Schweinfurthin A (SA). In addition to its anti-proliferative effects, SA caused dramatic reorganization of the actin cytoskeleton. When the NF1 Ras-regulatory domain was re-expressed in an Nf1-/- astrocytoma cell line derived from the NPcis model, these cells became resistant to the effects of SA. This led us to hypothesize that SA mediates its effects on the actin cytoskeleton and proliferation by phenocopying neurofibromin in its regulation of Rho and mitogenic signaling pathways. Our objective is to understand if and how this is the case.

To further that end, the goal of this study was to begin to probe the coordination of proliferation and cytoskeletal tension, as has been previously reported in other studies. We cultured individual SA- and DMSO-treated MPNST cells derived from the NPcis model onto pre-defined micropatterns of fibronectin. By constraining cell shape, quantitative structural properties of cells were revealed that were otherwise masked when cells were grown on standard coverslips. We observed dose-dependent increases in the concave curvature of cell edge segments not attached to the underlying fibronectin. Such curvature is known to represent decreased cell tension exerted by the actin cytoskeleton. Across the same SA doses, we measured a decrease in cyclin D1 and cyclin D3 total protein levels and a decrease in retinoblastoma protein phosphorylation, which was consistent with flow cytometry data showing a G1 blockade. Thus, we conclude that proliferation and cytoskeletal tension are coordinated in cells sensitive to the candidate drug, SA.

Funded by NCI Contract No. HHSN261200800001E.

Citation Format: Thomas Turbyville, Alla Brafman, De Chen, Kathryn Romanchuck, John Beutler, Karlyne Reilly, Stephen Lockett. Coordination of cell cycle repression and reduced cytoskeletal tension by the small molecule natural product, Schweinfurthin A. [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 3429. doi:10.1158/1538-7445.AM2013-3429
Abstract 3434: Inecalcitol, an analogue of 1,25D3, displays enhanced antitumor activity through the induction of apoptosis in a squamous cell carcinoma model system.
Authors: Ma Yingyu , Yu Wei-Dong , Hidalgo Alejandro A. , Luo Wei , Delansorne Remi , Johnson Candace S. , Trump Donald L.
Published: August 2014

Epidemiological data suggest an important role of vitamin D signaling in cancer progression, and experimental studies demonstrate that the active vitamin D metabolite 1α, 25-dihydroxyvitamin D3 (1,25D3) has broad spectrum anti-tumor activity. Hypercalcemia has often been suggested to limit the clinical application of these data. The 14-epi-analog of 1,25D3, inecalcitol (19-nor-14-epi-23-yne-1,25-(OH)2D3; TX522), was developed to have superagonistic anti-tumor activities but low hypercalcemia potential. We examined the anti-tumor activity of inecalcitol and the underlying mechanisms in a murine squamous cell carcinoma (SCC) model system. Compared to 1,25D3, inecalcitol showed enhanced vitamin D receptor (VDR)-mediated transcriptional activity. Inecalcitol suppressed SCC cell proliferation in a dose dependent manner with an IC50 value 30 times lower than that of 1,25D3. Both inecalcitol and 1,25D3 induced a comparable level of G0/G1 cell cycle arrest in SCC cells. The level of apoptosis induced by inecalcitol was markedly higher than that of 1,25D3. Apoptosis was mediated through the activation of the caspase 8/10- caspase 3 pathway. Further, inecalcitol markedly inhibited the mRNA and protein expression of c-IAP1 and XIAP compared to 1,25D3. In vivo, inecalcitol inhibits SCC tumor growth at 80 μg dose without hypercalcemia. Inecalcitol (320 μg) or 1,25D3 (0.312 μg) markedly inhibited tumor growth. Notably, inecalcitol induced a significantly higher level of apoptosis in the SCC xenograft model. We show that inecalcitol has potent anti-tumor activity in the SCC model system and this is associated with a stronger induction of apoptosis. These findings support the further development of inecalcitol in cancer treatment.

Citation Format: Yingyu Ma, Wei-Dong Yu, Alejandro A. Hidalgo, Wei Luo, Remi Delansorne, Candace S. Johnson, Donald L. Trump. Inecalcitol, an analogue of 1,25D3, displays enhanced antitumor activity through the induction of apoptosis in a squamous cell carcinoma model system. [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 3434. doi:10.1158/1538-7445.AM2013-3434