bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract C29: Bladder cancer-associated single nucleotide polymorphisms of vascular endothelial growth factor in 12 human cell lines
Authors: Lainesse Karine , Menard Sonia , Ke Danbing , Khan Salim A. , Prevost Gabrielle , Filion Mario C. , Phillips Nigel C.
Published: June 2012

Introduction: Vascular endothelial growth factor (VEGF), critical in angiogenesis, is important for solid tumor growth and metastasis. Three single nucleotide polymorphisms (SNP) in the promoter (rs833052, rs1109324 and rs1547651) and one in the 5′-UTR regions (rs25648) of VEGF have been associated with increased risk for bladder cancer, and one at intron 2 (rs3024994) with decreased risk in a large-scale case-control study. The genotype frequency of the five bladder cancer-associated SNPs among various ethnic groups is similar based on the dbSNP and the SNP500Cancer databases. The objective of this study was to determine the presence of these variants in a panel of 12 human bladder cancer cell lines.

Methods: Twelve bladder cancer cell lines, commonly used in urological research and derived from various ethnic groups, were included in the study. Genotypes of the five SNPs were determined for the12 cell lines by direct PCR sequencing. The basal VEGF secretion by these cell lines in culture was measured by ELISA using a microsphere-based array (Millipore) and Luminex® xMAP technology.

Results: The SNP genotype frequency was in agreement with that reported in the databases. The only bladder cancer-associated variant (rs833052: C>A) was detected in UM-UC-3, a cell line with many other genetic alterations. The secreted VEGF level in these cell lines as determined by ELISA varied considerably. No correlation between VEGF production and any particular SNP genotype could be established.

Conclusions: The reported bladder cancer associated variants in VEGF was detected in only one of the twelve cell lines. Though these SNPs are all located in the regulatory regions, it appears that they have no effect on basal VEGF production in the cultured cells. Considering the low prevalence of these variants reported, the role of these SNPs in VEGF production and bladder cancer remains to be defined.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C29.
Abstract C36: MBP-426, a novel liposome-encapsulated oxaliplatin, in combination with 5-FU/leucovorin (LV): Phase I results of a Phase I/II study in gastro-esophageal adenocarcinoma, with pharmacokinetics
Authors: Senzer Neil N. , Matsuno Kazuhiko , Yamagata Nobuyuki , Fujisawa Tadashi , Wasserman Ernesto , Sutherland William , Sharma Sunil , Phan Alexandria
Published: June 2012

Background: MPB-426 is a new transferrin-conjugated liposomal formulation of oxaliplatin, which can provide preferential tumor targeting and better bioavailability through the EPR effect and binding of transferrin receptors, respectively. In a recent phase I study, single agent MBP-426 given q3w showed thrombocytopenia as DLT, with 226 mg/m2 being the recommended phase II dose (RDP2). The current phase I–II study was designed to establish the RDP2 of MPB-426 in combination with 5-FU/LV (de Gramont schedule) and assess the efficacy of this combination in pretreated patients with gastro-esophageal (GE) adenocarcinoma.

Methods: MBP-426 IV was given q3w at two dose levels: 226 and 170 mg/m2 with fixed doses of 5-FU/LV. LV (400 mg/m2) was given concurrently with MBP-426 followed by IV bolus 5-FU (400 mg/m2), and then by 5-FU 2400 mg/m2 by continuous IV (CIV) infusion over 46 hours. The phase I dose assessment part was conducted in solid tumor patients. The phase II part will be limited to second line patients with gastric, gastro-esophageal or esophageal adenocarcinoma.

Results: The phase I part has been completed. Nine patients (pts) were included and treated. Median age: 61 years (55–67), gender M/F: 5/4, ECOG PS 0/1: 2/7, diagnosis: colorectal 4, pancreas 2, bladder 1, H&N 1 and cervix 1. Median number of prior lines: 4 (2–10). Four patients received a total of 11 cycles at the first dose level (MBP-426 226 mg/m2), in which two dose limiting toxicities (DLTs) were observed: grade 3 nausea and vomiting (1 pt) and grade 3 back pain (1 pt). Additional grade 3–4 toxicities were thrombocytopenia (3 pts), neutropenia (2 pts), transient rise of bilirubin (1 pt), and skin toxicity (1 pt with grade 3 erythema multiforme at cycle 4). To date, six patients (7 cy) have been explored at a lower dose level of 170 mg/m2. No DLTs were observed, establishing 170 mg/m2 as the RDP2. Grade 3–4 toxicity was limited to neutropenia in 1 pt. Oxaliplatin-induced peripheral neuropathy was not reported at either dose level, with 5 pts receiving an oxaliplatin equivalent dose ≥ 500 mg/m2. Efficacy: early data show 2 SD (4 cycles, 4+) and 1 PD (2 cycles) in 3 evaluable colorectal pts, all resistant to prior exposure to oxaliplatin. Pharmacokinetic data will be available for the meeting.

Conclusion: MBP-426 at 170 mg/m2 is the RDP2 for use in combination with 5-FU/LV (de Gramont schedule) every 3 weeks. Treatment at this dose was well tolerated. No peripheral neurotoxicity has been reported with this new formulation of oxaliplatin. Potential activity was observed in 2 oxaliplatin-resistant patients.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C36.
Abstract C38: Immunosuppression by indoleamine 2,3-dioxygenase in mesenchymal stem cells derived from adult human tissues
Authors: Lee Myoung Woo , Jang In Keun , Noh Yoo Hun , Kim Dae Seong , Kim Hye Ryung , Cheuh Hee Won , Lee Soo Hyun , Jung Hye Lim , Sung Ki Woong , Koo Hong Hoe
Published: June 2012

Mesenchymal stem cells (MSCs), which evoke only minimal immune reactivity, may have anti-inflammatory and immunomodulatory effects. In this study, we conducted a comparative analysis of the immunomodulatory properties of MSCs derived from adult human tissues including bone marrow (BM), adipose tissues (AT), umbilical cord blood (CB), and cord Wharton's jelly (WJ). Using a multiple cytokine detection assay, we showed that there were no significant differences in levels of secreted factors from non-stimulated MSCs. We compared the immunosuppressive effect of BM-MSCs, AT-MSCs, CB-MSCs, and WJ-MSCs on phytohemagglutinin (PHA)-induced T-cell proliferation. AT-MSCs, CB-MSCs, and WJ-MSCs effectively suppressed PHA-induced T-cell proliferation as effectively as did BM-MSCs. Levels of interferon (IFN)-γ secreted from activated T-cells increased over time, but these levels were significantly reduced when cocultured with each type of MSCs. In addition, the expression of hepatocyte growth factor, interleukin-10, transforming growth factor-β1, cyclooxygenase (COX)-1, and COX-2 were unchanged in MSCs treated with IFN-γ, while that of indoleamine 2,3-dioxygenase (IDO) increased. Use of an antagonist, 1-methyl-L-tryptophan, restored T-cell proliferation and confirmed an IDO contribution to IFN-γ-induced immunosuppression by MSCs. Addition of tryptophan catabolite, such as kynurenine, 3-hydroxykynurenine, or quinolinic acid, significantly decreased PHA-induced T-cell proliferation. These data indicate that IFN-γ produced by activated T-cells were correlated with induction of IDO expression by MSCs, which, in turn, suppressed T-cell proliferation through tryptophan depletion and the local accumulation of tryptophan catabolites. Our findings suggest that MSCs derived from AT, CB, or WJ could be substituted for BM-MSCs for treatment of allogeneic conflicts.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C38.
Abstract C43: Dasatinib resensitizes Kras mutant colorectal tumors to cetuximab
Authors: Dunn Emily F. , Iida Mari , Armstrong Eric , Wheeler Deric L.
Published: June 2012

Several recent clinical trials have demonstrated a strong correlation between Kras mutational status and resistance to cetuximab in metastatic colorectal cancer (mCRC). The work presented here sought to further understand the resistance of Kras mutant tumors to cetuximab and analyze dasatinib, a potent FDA approved SRC family kinase inhibitor, and its ability to overcome cetuximab resistance in this genetic setting. We analyzed 16 CRC lines for cetuximab growth response in vivo, expression of EGFR, SRC, and Kras mutational status. From this analysis we selected three Kras mutant lines (HCT116, LS180, LoVo), one Kras wild type line (SW48), and one EGFR negative line (Colo320DM) for further studies. Approximately two million cells from each line were inoculated in the dorsal flanks of athymic nude mice and allowed to grow to 100mm cubed. At that time mice were given 0.3mg of cetuximab twice weekly for 2 to 4 weeks. All Kras mutant lines were resistant to cetuximab in vivo, whereas the Kras wild type line showed significant tumor growth inhibition to cetuximab therapy. Each Kras mutant line was then analyzed for response to dasatinib alone. Mice were given 30mg/kg of dasatinib fives times weekly by oral gavage for 2 to 4 weeks. Results of these experiments indicated that Kras mutant tumors exhibited very little response to dasatinib monotherapy. However, dasatinib given in combination with cetuximab lead to significant tumor growth delay relative to vehicle controls. Collectively, these data suggest that Src and its family of kinases cooperate with the EGFR in the Kras mutant, mCRC setting and dual blockade has a distinct anti-tumor effect. These studies strengthen the rationale for the design of clinical trials combining dasatinib and cetuximab in the Kras mutant mCRC setting.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C43.
Abstract C54: In silico docking of the human cancer proteome in search of multitarget cancer therapeutics
Authors: Meroueh Samy , Li Liwei , Bum-Erdene Khuchtumur
Published: June 2012

A recent effort at uncovering the genome of various cancers has found multiple mutations across the genome of these cells reinforcing the view that cancer is a systems biology disease. The development of effective cancer therapeutics will require shifting the current paradigm from targeting individual proteins to instead targeting the broader signaling pathways. Towards that end, we have initiated an effort to employ in silico docking to screen against a large set of protein structures from the human proteome that have been implicated in cancer. We consider two datasets of proteins in our effort. First, a recently established dataset known as the Human Cancer Proteome Interaction Network (HCPIN) provides a set of structures corresponding to oncogenes as well as proteins that associate with them. Second, we created the human druggable protein (HDP) dataset that consists of targets of FDA-approved drugs. The surface of these 1,589 proteins was mapped for binding cavities that can accommodate a small molecule. We found more than 1,900 cavities spanning these proteins. We then prepared each of these binding cavities for in silico docking of 1,600 molecules from the NCI diversity set. The resulting 3,000,000 complexes were scored with multiple scoring functions to enable the ranking of molecules for each cavity. In an initial effort to identify anti-cancer therapeutics, we employed a combination of machine learning and fingerprint analysis to seek molecules whose binding profile would mimic the clinically used drug erlotinib. Among a set of 12 molecules that were selected, we found that four inhibited H1299 and H460 tumor cell growth in cell culture-based assays, with one molecule exhibiting an IC50 in the low micromolar region. We performed an assessment of the pharmacokinetic properties of these compounds to assess their suitability as potential leads for drug development.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C54.
Abstract C55: Efficacy of PI3K inhibitors to cancer cells with PIK3CA mutation or loss of PTEN expression
Authors: Dan Shingo , Sugita Hironobu , Seki Mariko , Mukai Yumiko , Yamazaki Kanami , Ishikawa Yuichi , Yamori Takao
Published: June 2012

Phosphoinositide 3-kinase (PI3K) pathway is frequently activated in cancer and is thought to be a promising therapeutic target. We previously reported a specific PI3K inhibitor ZSTK474 having a potent antitumor activity in vitro and in vivo without severe toxicity. Since then, diverse PI3K inhibitors have been developed, and some of them already entered clinical trials. Gain-of-function mutations of PIK3CA and loss of PTEN expression are frequently observed in various cancers and believed to activate PI3K-downstream effectors, but it is not well understood whether PI3K inhibitors have therapeutic efficacies against such cancers. In this study, taking advantage of pharmacologically well-characterized 39 human cancer cell lines (JFCR39), we examined the efficacies of eleven PI3K inhibitors including ZSTK474 and NVP-BEZ235, the mutational status and the protein expression levels of PIK3CA (p110) and PTEN in each of the cell lines. As a result, we found eight cell lines carrying missense mutations in PIK3CA. Most mutations were located in hotspot (E545K and H1047R), but some of them were in other residues. We evaluated kinase activities of these mutants and found that four of them including P449T (2.3-fold), E545K (8.5-fold), E545K/D549N (6.3-fold) and H1047R (5.8-fold) exhibited enhanced kinase activities compared to wild-type p110 , and seven of 39 cell lines had such mutation. On the other hand, mutational analysis of PTEN revealed that nine cell lines had missense mutation or deletion in PTEN, and seven of them did not express PTEN protein. Then, we compared the efficacies of PI3K inhibitors in cell lines with gain-of-function mutation of PIK3CA or loss of PTEN expression with those in other cell lines, and no significant correlation was observed. We further examined the inhibitory effect of three PI3K inhibitors on the enzymatic activity of p110 mutants, and all of three inhibited the kinase activity of E545K and H1047R mutant as well as wild-type p110 . These results suggested that PI3K inhibitors examined in this study were effective to cancer cells with PIK3CA mutation or PTEN loss equivalent to those having normal PIK3CA and PTEN expression, and that these PI3K inhibitors are applicable in clinical use for treatment of such cancers.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C55.
Abstract C59: Discovery and characterization of selective PI3Kα and PI3Kα/mTOR-dual inhibitors for targeting PI3Kα-activated tumors
Authors: Foster Paul G.
Published: June 2012

Background: The PI3K pathway regulates cell growth, proliferation and survival, and is dysregulated with high frequency in human tumors. PI3K pathway activation in tumors occurs via multiple mechanisms including prevalent mutation and amplification of the PIK3CA gene (which encodes the p110 subunit of PI3Kα). Downregulation of the lipid phosphatase PTEN also activates PI3K pathway signaling in a subset of tumors, via a mechanism reported to be PI3Kβ dependent. These findings suggest that isoformselective PI3K inhibitors may have utility in distinct, genetically defined tumor populations. Downstream of PI3K, mTOR controls cell growth and proliferation through its two distinct signaling complexes: mTORC1 and mTORC2. We report on the discovery of PI3Kα and dual PI3Kα/mTOR-selective inhibitors intended to target tumors harboring activating mutations or amplifications in PIK3CA.

Methods: We identified and optimized a series of PI3Kα and PI3Kα/mTOR inhibitors through high-throughput screening and medicinal chemistry techniques. Biochemical assays employed recombinant class 1 PI3K and mTOR proteins and cellular assays employed ELISA or western blot assays to monitor PI3K pathway signaling.

Results: We have identified potent (biochemical IC50s < 20 nM), ATP-competitive inhibitors of PI3Kα or PI3Kα and mTOR with excellent selectivity over other PI3K isoforms and a diverse panel of protein kinases. Cellular assays in the PI3Kα-activated MCF7 cell line demonstrate that these compounds inhibit phosphorylation of targets downstream of both PI3K (Akt) and mTOR (S6 and p70S6 kinase), and also inhibit tumor cell proliferation. However, in PC3 tumor cells lacking PTEN, effects on PI3K pathway readouts are less evident. In vivo pharmacodynamic analyses following oral administration of PI3Kα/mTOR inhibitors to mice bearing xenograft tumors demonstrate dose-dependent inhibition of phosphorylation of both PI3K and mTOR effectors at well-tolerated doses.

Conclusions: PI3Kα and PI3Kα/mTOR-selective small molecule inhibitors were discovered that demonstrate potent in vitro and cellular activity and good oral bioavailability. Cellular selectivity for PI3Kα mutant tumor cells versus PTEN deleted tumor cells was demonstrated. These compounds will allow an exploration of the efficacy and tolerability of selective PI3Kα and PI3Kα/mTOR inhibitors in preclinical tumor models.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C59.
Abstract C7: Sensitization of human colorectal cancer cells to TRAIL induced-apoptosis following inhibition of PI3K or HSP90
Authors: Saturno Grazia , Clarke Paul A. , Workman Paul
Published: June 2012

TRAIL (Tumor-Necrosis-Factor related apoptosis-inducing ligand) induces apoptosis by binding to the death receptors DR4 and DR5. Recombinant TRAIL or agonist antibodies against TRAIL death-receptors are now in Phase I/II clinical trials. Despite specificity for cancer cells, a percentage of tumor cells remain insensitive to TRAIL-induced apoptosis. Previous work has demonstrated that signaling through PI3K (phosphatidylinositol-3-kinase) can prevent TRAIL-induced apoptosis in different cancer cell types. Moreover, inhibition of HSP90 (Heat Shock Protein 90) by 17-AAG can sensitize colon cancer cells to TRAIL-induced apoptosis. The aim of this project is to explore the mechanism of TRAIL resistance and to focus on the role of PI3K and HSP90 signaling and the ability of PI3K or HSP90 inhibitors to reverse resistance to TRAIL-induced apoptosis in human colorectal cancer cells. We demonstrated that co-treatments with TRAIL and PI-103, a PI3K inhibitor, or 17-AAG, an HSP90 inhibitor, synergistically contributed to the apoptotic stimulus in TRAIL sensitive and resistant colorectal cancer cells through the amplification of both extrinsic and intrinsic apoptotic pathways; this effect was lower or not present in non-tumor cells, suggesting a therapeutic index. These data support the potential use of TRAIL in combination with PI3K or HSP90 inhibitors in patients with colorectal cancer who showed no response to the ligand alone.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C7.
Abstract C82: The role of the neuregulin family in the nucleus
Authors: Wang Ming
Published: June 2012

The neuregulin family consists of four members, NRG1- 4 each of which plays an important role in several normal physiological processes including tumorigenesis. We have focused on the 3 isoform of NRG1 which was found to localise to two subnuclear compartments: nucleoli and spliceosomes. We hypothesised that such localisation may have specific functions in modifying cell behaviour.

Methods: To examine whether nuclear accumulation of NRG1 occurs in other physiological conditions besides in ductal carcinomas of the breast, we immunohistochemically stained a range of normal and cancer tissues with antibodies to NRG1α or NRG1β. Second, Based on the previous observation that NRG1 3 localisation could change from the nucleolar pattern to the speckle pattern or vice versa, we monitored the trafficking of photoactivatable green fluorescent protein (PAGFP) tagged NRG1β3 in live cells using confocal fluorescence microscopy. We also explored the expression patterns of ErbB3 when co-expressed with NRG1β3 by immunofluorescence microscopy. To test the hypothesis whether nuclear targeting of NRG1β3 could be involved in stimulating cell mitogenesis we performed a Bromodeoxyuridine (BrdU) assay on cells transfected with NRG1β3 constructs.

Results: Nuclear expression of NRG1α and NRG1β was found in normal skin, adrenal and thyroid tissues and in various cancers. In general, NRG1α has a higher frequency of nuclear expression than NRG1β. By monitoring the PAGFP tagged NRG1β3 in live cells we observed that NRG1β3 moved from nucleoli to spliceosomes. As a cognate receptor of NRG1, ErbB3 was not colocalised with NRG1β3 in the nucleus. With respect to possible functions of intranuclear NRG1, the BrdU assay results showed that wildtype NRG1β3 and the mutant which lost the ability to target to nucleoli had no obvious effect on DNA synthesis.

Conclusion: Detection of nuclear expression of NRG1 in a range of normal and cancer tissues using immunohistochemical staining provides a physiological basis for further studies at the molecular level. The receptor-independent nuclear localisation of NRG1β3 suggests it may be participating in a novel-signalling pathway. Moreover, its association with nucleoli and spliceosomes, two nuclear compartments involved in ribosome synthesis and RNA splicing supports the hypothesis that nuclear NRG may have highly specific functions in defined nuclear compartments.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C82.
Abstract C89: Rac1 splicing variant Rac1b: Towards isoform selective inhibitors for the treatment of cancer
Authors: Laurent Desire , Casagrande Anne-Sophie , Leblond Bertrand , Beausoleil Eric , Taverne Thierry , Chauvignac Cedric , Pando Matt
Published: June 2012

The pivotal role of Rac1, a member of the Rho family of small GTPases, has been well characterized in the genesis of many cancers. The aberrant activation of Rac1 promotes uncontrolled proliferation, invasion and metastatic properties of human cancer cells.

Via ExonHit Therapeutics' alternative splicing discovery plateform DATAS™, we identified a repertoire of genes deregulated in cancer. Among them, Rac1b, an alternative splice variant of Rac1, was found deregulated and overexpressed in diverse cancer types, including breast and colon. Rac1b is constitutively active and transforming, and since it was shown to sustain cancer cell survival, such as for colorectal cancer cells, there is considerable interest in the development of small molecule inhibitors of Rac1/Rac1b.

We previously synthesized and fully characterized EHT 1864 as a pharmacological tool to study Rac1 functions(Shutes A et al. (2007). J Biol Chem. 282(49):35666-78). Then, using molecular docking algorithms and ExonHit's screening platform for nucleotide binding inhibitors, novel compounds were synthesized and characterized for Rac1/Rac1b inhibition and Rac1 versus Rac1b selectivity.

Here, we describe new Rac1/Rac1b inhibitors with better pharmacological profile than EHT 1864 and better potency in various in vitro cancer cell models. Some of these pharmacological tools are good candidates for subsequent in vivo cancer studies to study Rac1 and Rac1b mediated cellular functions and to modulate pathological conditions in which Rac1 or Rac1b deregulation may play a role.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C89.
Abstract C93: Novel monosubstituted oxaliplatin analogs with improved characteristics
Authors: Jungwirth Ute , Heffeter Petra , Gojo Johannes , Abramkin Sergey , Meelich Kristof , Galanski Michael , Körner Wilfried , Micksche Michael M. , Keppler Bernhard K. , Berger Walter
Published: June 2012

Background: Oxaliplatin has unique activity against colon cancer cells distinctly different from cisplatin. Aim of this study was to determine the anticancer properties of new mono-substituted oxaliplatin analogs, namely KP1537 and KP1691, to define the underlying cellular and molecular mechanisms.

Methods: Cytotoxic/antiproliferative effects were tested against selected human cancer cell lines by MTT analysis. The impact of the DNA damage response pathway on the drug activities were determined using HCT-116 human colon carcinoma cells together with p53 or p21/WAF1 deleted sublines. Furthermore, DNA damage, cell cycle distribution and induction of apoptosis were analysed by Western Blotting, FACS analysis and immunofluorescence. Platinum accumulation was determined by ICP-MS. For in vivo toxicity experiments, mice were treated with KP1691 and oxaliplatin.

Results: Both oxaliplatin analogues had IC50 values in the low µM range, with those for KP1691 being significantly lower in comparison to oxaliplatin. Additionally, KP1691 was less affected by the p53 status of the cells as compared to oxaliplatin. The new compounds were more rapidly taken up by the tumor cells and accumulated over time to higher intracellular concentrations as compared to oxaliplatin. In HCT-116 cells all platinum drugs inhibited the DNA synthesis in a p53-dependent manner, associated with a distinct induction of p53 and p21 in the HCT-116 p53 cell model. This suggests that inhibition of DNA replication due to platinum adducts leads to a p53-dependent cell cycle arrest. KP1537 and KP1691 induced potent G1/S and G2/M arrests in parental HCT-116. While at wild-type p53 conditions the platinum drugs resulted in almost complete loss of S-phase, this fraction was widely unchanged in the p53- deleted subline. Accordingly, expression of cellcycle related genes including cyclin A, cyclin B1, and CDK1 were down-regulated in a p53-dependent manner. Platinum drug-induced DNA damage was confirmed by the phosphorylation of H2AX. First in vivo toxicity experiments in mice indicated significantly less side effects (weight loss) after KP1691 than after oxaliplatin treatment.

Conclusion: Taken together, these findings suggest that KP1537 and KP1691 have promising anticancer properties in vitro. Several aspects, including the lower IC50 values, higher accumulation rates and less side effects of KP1537 and KP1691 as compared to the parental compound oxaliplatin, suggest them for further (pre-) clinical development.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C93.
Abstract C96: The effect of non-steroidal anti-inflammatory drugs (NSAIDs) in ovarian cancer cells
Authors: Valle Blanca L. , D'Souza Theresa , Becker Kevin G. , Wood William H. , Wersto Robert P. , Morin Patrice J.
Published: June 2012

Epidemiological and laboratory studies have suggested a protective role for non-steroidal anti-inflammatory drugs (NSAIDs) against various neoplasms, including ovarian cancer. In an attempt to clarify the mechanisms and molecular targets of tumor prevention by NSAIDs, we have studied the effects of multiple NSAIDs in several ovarian cancer cell lines. NSAID treatment decreased the viability of all the ovarian cancer cell lines examined. To determine the effect of NSAIDs on the cell cycle, flow cytometry analysis was performed. Cells treated with indomethacin, ibuprofen or celecoxib were arrested in G1 phase, while cells treated with diclofenac underwent an S/G2 arrest. In addition, induction of apoptosis was cell line specific. The levels of several cell cycle regulatory and apoptosis proteins were examined. The levels of phosphorylated Rb protein decreased and phospho-p53 increased upon treatment with NSAIDs, suggesting an involvement of these proteins in the response of ovarian cancer cells to NSAID treatment. In order to further clarify the mechanisms of action of NSAIDs in ovarian cancer cells, gene expression profiling experiments were performed. Ovarian cancer cell lines Hey, OVCAR5, and UCI101 were treated with diclofenac, indomethacin, celecoxib, and ibuprofen, and microarray analysis was performed. A large number of gene expression changes were observed in the different cell lines treated with various NSAIDs. Among the most consistent and reproducible changes observed, S-phase kinase associated protein 2 (Skp2) was found to be downregulated at the mRNA and protein levels in all cell lines with all NSAIDs tested. In addition, Damage inducible transcript-4 (DDIT4) was found to be upregulated in all cell lines upon treatment with diclofenac, indomethacin and celecoxib. Skp2 forms part of the SCF ubiquitin ligase and is involved in the targeting for degradation of several proteins, including the cyclin dependent kinase inhibitors p21 and p27, therefore facilitating S-phase entry. DDIT-4, is transcriptionally activated upon p53 activation and it is known to inhibit mTOR signaling. In conclusion, we have identified a number of target genes that may be important for the NSAID-induced cell cycle arrest and apoptosis and their exact roles are currently being investigated.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C96.
Abstract PR-10: RNAi-directed identification of chemosensitizers of GSK923295 response
Authors: Yin Holly , Kiefer Jeff , Chow Donald , Que Quick Q. , Dastrup Erica , Weber Barbara , Wooster Richard , Mousses Spyro
Published: June 2012

Centromere-associated protein E (CENP-E) is a kinesin-like motor protein that plays a central role in chromosome segregation and control of cell division. GSK923295 (GSK295) is a potent and selective small-molecule inhibitor of human CENP-E ATPase activity, and has demonstrated broadspectrum activity in vivo against human tumor xenografts. GSK295 is currently being evaluated as a novel anticancer drug in Phase I clinical trials. RNAi phenotype profiling in the Druggable Genome was applied to discover specific genes that regulate GSK295 sensitivity. This study focused on nonsmall cell lung carcinoma (NSCLC) with three cell lines evaluated for HT-RNAi drug sensitivity analysis. A robust HT-RNAi cell-screening assay for GSK295 sensitivity was successfully developed for all cell lines. The sensitization assay was developed as following: cells were reversely transfected with individual siRNA for 24hr followed by the treatment of various concentrations of GSK295 for 72hr when cell viability was measured. Among 3 optimized cell lines, the A427 cell line, which appeared to be relatively more resistant to GSK295, was selected for a primary RNAi screen against 14,000 siRNA sequences covering 7,000 gene targets (Druggable Genome). Primary screen data was rigorously evaluated for multiple quality control metrics and found to exceed all expected performance parameters with >98% global transfection efficiency, <0.25 CV values, and minimal plate-to-plate and set-to-set variations observed. The top 10% drug sensitization hits were chosen, of which 240 of them were confirmed in subsequent analysis with the A427 cell line. To further validate these sensitizers to GSK295 and to eliminate the off-target effects, we compared the response of these sensitizers (4 unique sequences per gene target were selected) to GSK295 in two other NSCLC cell lines (A549 and NCI-H460) and selected the validated hits that have two or more unique siRNA sequences per gene target demonstrating sensitization to GSK295 response. Additionally, 23 chemical compounds targeting 16 of the top validated RNAi hits from the screen were tested for synergistic combination with GSK295. Notable synergism with GSK295 was observed with 10 inhibitors against 7 validated RNAi gene targets. In conclusion, the results yielded multiple sensitizing gene targets, with a high correlation between the targets that showed RNAi sensitization as well as synergy in combination with small molecules that inhibit the same targets. These new targets represent candidate GSK923295 sensitizing targets that warrant further investigation to support the development of rational GSK923295 combinations to maximize therapeutic efficacy in the clinic.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):PR-10.
Abstract PR-3: Antitumor efficacy on glioma models of the dual CDK/TRKA inhibitor PHA-848125, able to cross the blood brain barrier
Authors: Albanese Clara , Pastori Wilma , Locatelli Giuseppe , Alzani Rachele , Fiorentini Francesco , Festuccia Claudio , Pesenti Enrico , Galvani Arturo , Isacchi Antonella , Mercurio Ciro , Brasca Maria Gabriella , Ciomei Marina
Published: June 2012

Malignant gliomas, the most common primary brain tumors, are highly invasive and neurologically destructive tumors with very poor prognosis due to the impossibility of removing tumor tissue completely by surgery and the limited activity of therapeutic agents.

PHA-848125 is a dual CDKs and TRKA inhibitor and is therefore potentially active against two major pathways involved in glioma pathogenesis: signaling mediated by tyrosine kinase growth factor receptors and control of G1-S phase progression of the cell cycle.

PHA-848125 has demonstrated good antitumor activity in many preclinical tumor models, good tolerability in Phase 1 studies and it is currently undergoing Phase 2 clinical trials.

To explore the rationale for pursuing brain tumors as a clinical indication for PHA-848125, we firstly analyzed distribution of the 14C-labeled compound, demonstrating that it is able to efficiently cross the blood brain barrier. Next, we tested in vitro activity of the compound on a panel of glioma cell lines showing that it was able to inhibit cell proliferation (IC50: 1.4–2.5 µM), DNA synthesis (inhibition of BrdU incorporation ranging from 39 to 92 %), cell cycle markers (inhibition of Phospho-pRb, cdc6, cyclin A and cyclin B1 expression) and signal transduction markers (inhibition of Phospho-TRKA, Phospho-MAPK and Phospho-AKT). Significantly, it was also able to induce cell death through autophagy (mainly in cells with functional Retinoblastoma protein) or apoptosis (in cells with disrupted Retinoblastoma pathway). Antitumor efficacy in vivo was evaluated both in subcutaneous and intracranially implanted tumors. PHA-848125 showed significant antitumor efficacy by oral route: tumor growth inhibition of 71% and 80% on U87MG and U251 subcutaneous xenograft models, respectively and a 30% increase in survival time in the intracranial U251 model. Finally, in the U87MG xenograft model, the combination of PHA-848125 with temozolomide resulted in a synergistic effect and a clear therapeutic gain was also observed with a triple treatment adding PHA-848125 to radiotherapy and temozolomide. In conclusion, preclinical data obtained to date suggest that PHA-848125 may become a useful agent in chemotherapy regimens for glioma patients and support its evaluation in Phase 2 trials for this indication.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):PR-3.
Abstract A120: Morphological assessment of apoptosis in HeLa cervical cancer cell line by Dox-loaded magnetic nanocluster.
Authors: Kumar Sunil , Daverey Amita , Bhahdur Dhirendra
Published: June 2012

A facile one step procedure to prepare amine functionalized MgFe2O4 nanocluster of size less than 50nm in a simple solvothermal reaction using sodium borohydride as reducing agent has been developed. The Amine group was confirmed by FTIR and its magnetization at room temperature were found to be 38 emu/g. The specific absorption rate (SAR) of the magnesium-ferrites nano-cluster were found to be 45.38 and 153.98 W/g of Fe with an applied field of 7.64 and 10.0 kA/m respectively at fixed frequency of 425kHz. Succinic anhydride were used to coat the nano-particle to load the Doxorubicin. The loading efficiency of was determined to be 79.4%. The high loading efficiency of Dox was observed due to porous nature of particle which was confirmed by Nitrogen adsorption-desorption isotherm. MTS assay results indicated that no significant cytotoxicity effect was detected with MgFe2O4 nano-cluster at concentration upto 0.4mg/ml after 24 and 48h of incubation with L929 cells. At 24h and 48h, the IC50 value of the Dox-MgFe2O4 nano-conjugates against HeLa cells was observed 3.5 and 1 μg/ml respectively. After 48 h of incubation of Dox loaded MgFe2O4 nano-cluster with HeLa cells, almost 99% cells were undergone apoptosis, which was observed by Confocal microscope. In conclusion MgFe2O4 nano-cluster have a potential for synergistically combine the effect of two therapeutics modalities chemotherapy and hyperthermia to induce apoptosis in HeLa cancer cell.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A120.
Abstract A128: Dual properties of fluorescence probe and anticancer agent of small molecules developed for cancer research.
Authors: Chang TaChau , Huang Wei-Chun , Wang Zi-Fu , Cho Chih-Chien
Published: June 2012

The dual properties of fluorescence probe for cancer diagnosis and G-quadruplex (G4) stabilizer for cancer treatment of 3,6-bis(1-methyl-4-vinylpyridinium)carbazole diiodide (BMVC) [1,2] inspire us to develop second-generation BMVC derivatives. A number of distinct BMVC derivatives were synthesized to investigate the correlation between cellular response and biological function of BMVC derivatives for cancer treatment. The fluorescence properties of BMVC derivatives allow us to visualize the cellular localization and possibly monitor the drug-target interaction. Together with their biological and toxicological analyzes, the correlation between cellular response and biological function of BMVC derivatives may allow us to elucidate their therapeutic mechanisms for cancer treatments.

Here we have evaluated the efficacy of two BMVC derivatives, 3,6-bis(1-methyl-4-vinylpyridinium iodide)-9-(1-(1-methyl-piperidinium iodide)-3,6,9-trioxaundecane) carbazole (BMVC-8C3O) and 3,6-bis(1-methyl-4-vinylpyridinium iodide)-9-(1-(1-methyl-piperidinium iodide) dodecyl) carbazole (BMVC-12C), in CL1-0 cancer treatment. Both molecules are mainly detected in lysosome of MRC-5 normal cells. However, BMVC-12C is mainly accumulated in mitochondria of cancer cells, while BMVC-8C3O is distributed in nucleus and mitochondria of cancer cells. Although BMVC-8C3O is a better G4 stabilizer than BMVC-12C to human telomeres, BMVC-12C inhibits population doubling of CL1-0 cancer cells earlier than BMVC-8C3O. Apparently, they have different therapeutic mechanisms. Fluorescence imaging showed competition of BMVC-12C with Mitotracker red into mitochondria, implying that BMVC-12C probably accumulates into mitochondria inner membrane. The accumulation of BMVC-12C in mitochondria induces cytochrome C release and cell death. In addition, CL1-0 cancer cells treated by BMVC-12C for 72 h will lose mitochondria membrane potential and increase the level of reactive oxygen species. Our results suggest that BMVC-12C is a novel mitochondriotoxic molecule.

References:

1. L.J. Liao, et al. Analyst 134, 708 (2009).

2. F.Z. Huang, et al. Mol. Cancer Res. 6, 955 (2008).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A128.
Abstract A160: Recognition of UbcH5c and the nucleosome by the Bmi1/Ring1b ubiquitin ligase complex.
Authors: Bentley Matthew L. , Corn Jacob E. , Dong Ken C. , Phung Qui , Cheung Tommy K. , Cochran Andrea G.
Published: June 2012

Background and Objectives: The Polycomb repressive complex 1 (PRC1) mediates gene silencing, in part by monoubiquitination of histone H2A on lysine 119 (uH2A). Bmi1 and Ring1b are critical components of PRC1 that heterodimerize via their N-terminal RING domains to form an active E3 ubiquitin ligase. A longstanding question in this field is how a single lysine residue in the nucleosome is singled out for ubiquitin modification and why only one ubiquitin is added rather than a chain of ubiquitins. To better understand how Bmi1/Ring1b controls monoubiquitination of H2A, we structurally characterized the E2 and nucleosome interaction sites on Bmi1/Ring1b.

Methods: X-ray crystallography was used to determine the structure of a complex between Bmi1/Ring1b and UbcH5c, its cognate E2 enzyme. Fluorescence-polarization assays were used to measure the binding of Bmi1/Ring1b to a synthetic DNA duplex. Site-directed mutagenesis was used to assess the importance of residues at the potential Bmi1/Ring1b-DNA interface, and in vitro ubiquitin ligase assays were used to measure the catalytic activity of mutant complexes. Molecular modeling studies were performed using HADDOCK v2.0.

Results: We have determined the crystal structure of a complex between the Bmi1/Ring1b RING-RING heterodimer and the E2 enzyme UbcH5c and find that UbcH5c interacts with Ring1b only, in a manner fairly typical of E2-E3 interactions. However, we further show that the Bmi1/Ring1b RING domains bind directly to duplex DNA through a basic surface patch unique to the Bmi1/Ring1b RING-RING dimer. Mutation of residues on this interaction surface leads to a loss of H2A ubiquitination activity. Through site-directed mutagenesis we have been able to uncouple the E2 binding and the DNA binding activities of Bmi1/Ring1b, indicating that Bmi1/Ring1b uses distinct binding surfaces to recognize E2 and the nucleosomal substrate. Computational modeling of the interface between Bmi1/Ring1b-UbcH5c and the nucleosome suggests that Bmi1/Ring1b interacts with both nucleosomal DNA and an acidic patch on histone H4 to achieve specific mono-ubiquitination of H2A.

Conclusions: Our data show that the direct interaction of the RING domains with nucleosomal DNA is crucial for the ubiquitin ligase activity of Bmi1/Ring1b. To our knowledge, this is the first example of a RING-domain E3 ligase binding directly to its substrate via the RING domain. Our results point to a novel mechanism of substrate recognition, and control of product formation, by Bmi1/Ring1b.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A160.
Abstract A165: mTOR kinase inhibition causes feedback-dependent biphasic regulation of AKT signaling.
Authors: Rodrik-Outmezguine Vanessa S. , Chandarlapaty Sarat , Pagano Nen C. , Poulikakos Poulikos I. , Scaltriti Maurizio , Moskatel Elizabeth , Baselga Jose , Guichard Sylvie , Rosen Neal
Published: June 2012

mTORC1 inhibitors relieve feedback inhibition of receptor tyrosine kinases (RTKs) and cause mTORC2-dependent phosphorylation of AKT S473 and activation of AKT kinase and signaling. mTOR kinase inhibitors block mTORC1 and mTORC2 and thus do not cause the mTORC2 activation of AKT observed with the mTORC1 inhibitor rapamycin. We now show, however, that these drugs have a biphasic effect on AKT. Inhibition of mTORC2 leads to AKT S473 dephosphorylation and a rapid but transient inhibition of AKT T308 phosphorylation and AKT signaling. However, inhibition of mTOR kinase also relieves feedback inhibition of RTKs leading to subsequent PI3K activation and rephosphorylation of AKT T308 sufficient to reactivate AKT activity and signaling. Thus, catalytic inhibition of mTOR kinase leads to a new steady state characterized by profound inhibition of mTORC1 and accumulation of activated AKT phosphorylated on T308 but not S473. Combined inhibition of mTOR kinase and the induced RTKs fully abolishes AKT signaling and results in profound cell death and tumor regression in vivo. These results reveal the adaptive capabilities of oncogenic signaling networks, as AKT signaling may be driven by a loss-of-feedback-induced AKT species lacking S473 phosphorylation. The addition of RTK inhibitors can prevent this reactivation of AKT signaling and cause profound cell death and tumor regressions in vivo, highlighting the possible need for combinatorial approaches to block feedback-regulated pathways.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A165.
Abstract A184: Integrative molecular and functional profiling of HER2 amplified breast cancers to identify new gene targets.
Authors: Shiu Kai-Keen , Wetterskog Daniel , Mackay Alan , Natrajan Rachael , Lambros Maryou , Sims David , Bajrami Ilirjana , Brough Rachel , Frankum Jessica , Horlings Hugo , van der Vijver Marc , Turner Nicholas , Reis-Filho Jorge , Lord Christopher , Ashworth Alan
Published: June 2012

Up to 15% of all breast cancers are defined as HER2+ve where overexpression is due to amplification of the HER2 oncogene. Treatment of patients with these clinically aggressive tumors has been dramatically improved by the use of trastuzumab and lapatinib. However a significant proportion of patients' tumors remain resistant to these therapies. Therefore new biomarkers and additional therapeutic targets are urgently required for what remains a clinically challenging and biologically heterogeneous disease.

We describe an integrated genetic, transcriptomic and functional analysis of a wide panel of genes amplified and overexpressed in HER2 amplified breast tumors to identify new gene targets. Molecular profiling of 45 sporadic primary HER2 amplified breast cancers, 14 commercially available HER2 amplified and 9 non HER2 amplified breast cancer cell lines was performed. Genome wide copy number and transcriptomic array data were integrated and identified 511 genes that were significantly overexpressed when amplified in the tumors. The functional significance of 369 of these genes, which were recurrently amplified in the cell lines, was examined using parallel high throughput siRNA screens.

Multiple genetic dependencies were observed. We demonstrate that loss of cell viability following silencing of HER2 by multiple siRNAs significantly correlated with sensitivity to lapatinib and other HER2 targeted agents. We also show that silencing of TFAP2C caused significant loss of cell viability when amplified and overexpressed in multiple HER2 amplified breast cancer cell lines. Down regulation of HER2 expression and cell apoptosis was also observed. The potential clinical value of TFAP2C overexpression for patients with HER2+ve breast cancer is currently being explored in adjuvant HER2+ breast cancer trials of chemotherapy +/− HER2 targeted agents. We propose that TFAP2C amplification and overexpression represents a genetic dependency in HER2+ breast cancer and is a clinically relevant candidate biomarker.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A184.