bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract C113: p21 downregulation is an important component of PAX3/FKHR oncogenicity and its reactivation by histone deacetylase inhibitors enhances combination treatment
Authors: Hecker Regina , Amstutz Ralf A. , Wachtel Marco , Walter Dagmar , Niggli Felix K. , Schafer Beat W.
Published: June 2012

A number of drugs developed against cancer-specific molecular targets have been shown to offer survival benefits alone or in combination with standard treatments, especially for those cases where tumor pathogenesis is dominated by a single molecular abnormality. One example for such a tumor type is alveolar rhabdomyosarcoma (aRMS) which is characterized by a specific translocation creating the oncogenic PAX3/FKHR transcription factor, believed to be the main molecular basis of the disease. Recently, we were able to demonstrate that the small molecule inhibitor PKC412 exhibits strong antitumor activity against aRMS by reducing the transcriptional activity of PAX3/FKHR.

In the present study, we screened for combination strategies that are superior to PKC412 treatment alone. Interestingly, we found that the combination of PKC412 with histone deacetylase (HDAC) inhibitors like valproic acid (VPA) synergistically induced apoptosis resulting in suppressed aRMS tumor growth in vivo.

We provide evidence that the enhanced antitumor effect upon combination treatment is achieved by VPA-induced reactivation of p21 which is downregulated in aRMS tumor cells via destabilization of its transcriptional regulator EGR1 by PAX3/FKHR. This identifies downregulation of p21 as a crucial event in PAX3/FKHR oncogenicity. Our study not only highlights a possible mechanism behind the increased efficacy of combination treatment but also indicates that different levels of PAX3/FKHR oncogenicity can be exploited therapeutically by a specific combination of anticancer drugs to increase their therapeutic potential.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C113.
Abstract C118: Influence of CYP2D6 phenotype on tamoxifen-treatment outcome in women with metastatic breast cancer
Authors: Lammers Laureen A. , Mathijssen Ron H.J. , van Gelder Teun , Bijl Monique J. , de Graan Anne-Joy M. , Seynaeve Caroline , van Fessem Marianne , Berns Els M. , Vulto Arnold G. , van Schaik Ron H.
Published: June 2012

Introduction: Cytochrome P450 2D6 (CYP2D6) plays a crucial role in the metabolism of tamoxifen into the active metabolite endoxifen. Genetic variation in the CYP2D6 gene and co-administration of CYP2D6 inhibiting medication markedly reduce endoxifen plasma concentrations in patients. The impact hereof on the outcome of tamoxifen therapy in metastatic breast cancer is insufficiently known. In this cohort study, we examined the effect of CYP2D6 predicted phenotype (the combined effect of genetic variants and concomitant use of CYP2D6 inhibiting medication) on time to progression (TTP) and overall survival (OS) in women using tamoxifen for metastatic breast cancer was examined.

Methods: We selected patients treated with tamoxifen (40mg/day) for hormone receptor positive metastatic breast cancer of whom blood samples for pharmacogenetic analysis (CYP2D6*3,*4,*5,*6) were available. Patient charts were reviewed to assess patient and tumor characteristics, TTP, OS, and use of CYP2D6 inhibitors.

Results: Patients with poor CYP2D6 metabolizer phenotype (PMs) had a worse OS compared to extensive metabolizers (EMs) (HR = 2.11; P=0.031; 95% CI: 1.07–4.15, see Table). Patients with an intermediate (IM) and extensive metabolizer (EM) phenotype had comparable TTP and OS, and were further considered as one group in the Kaplan-Meier estimates. For PMs, the Kaplan-Meier estimate demonstrated a shorter TTP compared to the combined group of IMs and EMs: 1.7 years (95% CI: 0.9–2.5) versus 2.9 years (95% CI: 2.3 – 3.6) respectively (P = 0.089). Overall survival of the group of IM/EM patients was significantly longer than for PM patients, being 9.9 years (95% CI: 8.2–11.6) versus 5.4 years (95% CI: 3.7–7.1), respectively (P=0.012). Co-administration of CYP2D6 inhibitors alone was also associated with a worse OS (HR=3.55; P=0.002; 95% CI: 1.59–7.96) and a worse TTP (HR=2.97; P=0.008; 95% CI: 1.33–6.67) compared to patients without CYP2D6 inhibitors.

Conclusions: CYP2D6 phenotype is an important predictor of treatment outcome in women treated with tamoxifen for hormone receptor positive metastatic breast cancer. Co-administration of CYP2D6 inhibitors worsens outcome of tamoxifen therapy, and should therefore be discouraged.

Effect of CYP2D6 phenotype on TTP and OS CYP2D6 phenotype Time to progression (TTP) Overall Survival(OS) Cases (n=99) Cases (n=99) HR (95% CI) HR (95% CI) P-value P-value EM n=50 n=33 HR=1.00 (ref) HR=1.00 (ref) IM n=36 n=22 HR=1.05 (0.67–1.65) HR=0.88 (0.51–1.53) P=0.82 P=0.66 PM n=13 n=12 HR=1.74 (0.92–3.28) HR=2.11 (1.07–4.15) P=0.09 P=0.03

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C118.
Abstract C12: Preclinical characterization of a novel FLIP-targeted antisense phosphorothioate oligonucleotide
Authors: Logan Andrew E. , Wilson Timothy R. , Johnston Patrick G. , Longley Daniel B.
Published: June 2012

FLIP (FLICE-inhibitory protein) is a major inhibitor of death receptor-mediated activation of caspase 8 (FLICE, Fas-associated death domain-like interleukin-1 -converting enzyme). We have previously found that FLIP is not only a key regulator of death receptor-mediated apoptosis, but also inhibits chemotherapy-induced apoptosis in several cancers. Furthermore, we found that silencing FLIP expression using siRNA approaches was in itself sufficient to induce apoptosis in several different cancer models. These results suggest that FLIP is a promising therapeutic target in several cancers. In this study, we describe the pre-clinical characterisation of a novel FLIP-targeted antisense phosphorothioate oligonucleotide (AS PTO). Of 5 potential sequences identified, one AS PTO potently down-regulated FLIP. Down-regulation of FLIP by this AS PTO resulted in caspase 8 activation and apoptosis induction in non-small cell lung (NSCLC), colorectal and prostate cancer cell lines. Furthermore, the FLIP AS PTO sensitized cancer cells to apoptosis induced by the clinically relevant death ligand TRAIL (TNF-related apoptosis-inducing ligand). Moreover, the FLIP AS PTO also sensitized NSCLC cells to apoptosis induced by the chemotherapeutic agents cisplatin and taxol. Importantly, compared to a control non-targeted PTO, the FLIP-targeted AS PTO demonstrated in vivo antitumor activity against NSCLC xenografts and enhanced the antitumor effects of cisplatin. In summary, we have identified a novel FLIP-targeted AS PTO that has in vitro and in vivo activity and which may therefore have potential for clinical development.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C12.
Abstract C126: Exposure-response (E-R) relationship in ABT-263-induced thrombocytopenia and the effect of platelet transfusion in dogs
Authors: Xiong Hao , Noertersheuser Peter A. , Pradhan Rajendra S. , Mittelstadt Scott W. , Marsh Kennan C. , Humerickhouse Rod A. , Awni Walid M.
Published: June 2012

Background: Thrombocytopenia is the most prominent dose-limiting toxicity in animals and humans following ABT-263 administration. ABT-263 induces apoptosis, rather than lysis, of circulating platelets resulting in premature platelet senescence. This differs from typical chemotherapy induced thrombocytopenia, which is related to myelosuppression. Bcl-XL is believed to play a key role in the thrombocytopenia induced by ABT-263. Platelet transfusion has been widely adopted in clinical practice when platelet count drops below 10,000/ L. The effect of platelet transfusions on circulating platelet levels have been examined in four studies in dogs.

Methods: Pharmacokinetic (PK) and pharmacodynamic (PD) data from a total of 76 dogs from the four studies were analyzed sequentially. First, ABT-263 plasma concentration data in dogs were described using a two-compartment model with first-order absorption (for oral dosing only) and elimination. Then, the time courses of circulating platelet levels were described using a semi-physiological model. The model includes a progenitor compartment for proliferating blood cells linked to a series of three compartments representing the maturation chain in the bone marrow and the central circulation compartment. A feedback system is included in the model to allow the system to respond to disturbances in the platelet concentrations. It is assumed that ABT-263 increases the degradation rate of circulating platelets in a concentration-dependent manner.

Results: The medians of the E-R model parameters were comparable among the studies, indicating that the E-R relationships between ABT-263 concentrations and platelet response are consistent across all four studies in dogs. Simulations using the model further supported that platelet transfusion is an effective rescue method for low platelet counts caused by ABT-263 treatment both at clinically-relevant ABT-263 concentrations (∼4 g/mL) and at the higher ABT-263 concentrations (7 g/mL) in dogs. The effects of platelet transfusion were smaller and more transient when the transfusion was administered at higher ABT-263 concentrations (7 g/mL) compared to those administered at lower ABT-263 concentrations (∼4 g/mL). Multiple transfusions are expected to produce larger and more prolonged effects on circulating platelet levels than a single transfusion.

Conclusion: Thrombocytopenia induced by ABT-263 in dogs is dependent on ABT-263 concentrations and could be well described by a semi-physiological E-R model. Platelet transfusion should be started after ABT-263 concentration has dropped and multiple transfusions are needed to produce larger and more prolonged effects on circulating platelet levels.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C126.
Abstract C133: Effect of casopitant, a novel neurokinin-1 receptor antagonist, on the pharmacokinetics (PK) of the oral contraceptive pill (OCP)
Authors: Adams Laurel M. , Zamuner Stefano , Fina Paolo , Peroni Michela , Johnson Brendan M. , Fernandes Sofia
Published: June 2012

Background: Clinical studies suggest that casopitant, a novel neurokinin-1 receptor antagonist in development for prevention of postoperative and chemotherapy-induced nausea and vomiting (PONV and CINV, respectively), is a mild to moderate inhibitor of CYP3A4. However, in cultured human hepatocytes, induction of CYP3A4 and UGT enzymes also has been observed. Since casopitant is intended for use in women receiving moderately emetogenic chemotherapy for breast cancer, the current studies evaluated the pharmacokinetics (PK) of a combined monophasic oral contraceptive pill (OCP) coadministered with repeat-dose casopitant.

Methods: Healthy female subjects of non-childbearing or of childbearing potential taking OCP were enrolled. Study 1 (N=11) was a randomized, double-blind, placebo-controlled, repeat-dose, crossover study. OCP containing 30 µg ethinylestradiol (ETE) and 150 µg levonorgestrel (LEV) was administered for 2 complete menstrual cycles (21 days + 7-day washout), plus oral placebo or 60 mg casopitant on days 1–14 of each cycle. Study 2 (N=61) was a single-blind, placebo-controlled, repeat-dose, single-sequence study. OCP was administered for 2 complete cycles as above with oral casopitant (30 mg or 120 mg) or placebo administered for cycles 1 and 2, respectively. Casopitant or placebo was administered for 28 consecutive days, beginning 7 days before OCP administration. The geometric least squares mean ratios and 90% confidence intervals (CI) for ETE and LEV AUC and Cmax were calculated to compare OCP + casopitant vs OCP + placebo.

Results: In study 1, ETE AUC(0−) and Cmax were decreased on average by 16% and 17%, respectively, when the OCP was coadministered with 60 mg casopitant vs placebo. Geometric mean LEV AUC(0−) increased 4%, and Cmax decreased 17%. In study 2, for ETE, OCP + casopitant decreased AUC(0−) on average by 15% and 40%, and Cmax by 24% and 32% with 30 mg and 120 mg, respectively. For LEV, the largest mean change in exposure was a 13% decrease in Cmax with 120 mg casopitant given daily for 14 days. Coadministration of casopitant with the combined OCP was well tolerated in both studies.

Conclusions: Continuous coadministration of casopitant for 14 or 28 days with a combined monophasic OCP resulted in only a small change in LEV exposure, but a dose-dependent reduction in ETE exposure that may reduce the efficacy of hormonal contraceptives. The effect of a single oral dose of casopitant on hormonal contraceptives has not been studied.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C133.
Abstract C137: Pinpointing a protein-protein interaction site of focal adhesion kinase and VEGFR-3 suppresses cancer growth in vivo
Authors: Kurenova Elena V. , Hunt Darrell , Ucar Deniz , He Dihua , Nyberg Carl , Hochwald Steven , Magis Andrew , Ostrov David , Cance William G.
Published: June 2012

Tumor cell survival depends on activation of signaling pathways that suppress the apoptotic stimuli of invasion and metastasis. FAK and VEGFR-3 are tyrosine kinases that have been identified as critical signaling molecules for these processes. Previously we have shown that VEGFR-3 and FAK physically interact and are overexpressed in cancer cells to provide a significant survival advantage for the tumor cells. We subsequently identified a novel small molecule inhibitor that targeted VEGFR-3-FAK site of interaction and disrupted the survival function of these two proteins. We utilized the crystal structure of the FAK focal adhesion targeting (FAT) domain for molecular docking of small molecules that targeted the VEGFR-3 binding site on FAK. We identified a small molecule C4 (Chloropyramine hydrochloride), that disrupted VEGFR-3-FAK binding, and abrogated the phosphorylation of VEGFR-3 while reducing the phosphorylation of FAK. In vitro testing of this compound resulted in the selective growth inhibition, reduction in motility and invasion, and induction of apoptosis in a time- and dose-dependent manner in many cancer cell lines, especially those that overexpressed VEGFR-3. In vivo, C4 showed a marked reduction of tumor growth and was synergistic with doxorubicin chemotherapy in breast cancer xenograft models, with dacarbazine in melanoma xenograft models, and with gemcitabine in pancreatic cancer xenograft models. These results demonstrate that targeting the FAK-VEGFR-3 interaction with a small molecules inhibited the survival function of these two tyrosine kinases, representing a unique approach for molecular-targeted highly-specific cancer therapeutics.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C137.
Abstract C145: The nonredundant oncogenic role of miR-17-5p in gastric cancer in vitro
Authors: Lee Seok Cheol , Kim Chang Hee , Green Jeff , Kim Hark Kyun
Published: June 2012

MicroRNAs are non-coding RNA molecules that consist of 19 to 25 nucleotides. Overexpression of miR-17-92 cluster is reported in many human cancers including gastric cancers, the second most common cause of cancer mortality worldwide. While the data in the literature predominantly supports the oncogenic role of miR-17-92 cluster, some studies suggested tumor suppressor role of the miR-17-92 cluster. Given that function of a given microRNA is context-dependent, the exact function of miR-17-92 cluster in gastric cancer cells should be experimentally documented in order to validate this microRNA as a novel therapeutic target. MiR-17-5p is one member of the miR-17-92 cluster, the other members of which are miR-18a, miR-19a, miR-20a, miR-19b and miR-92. Although knockdown of mir-17-5p is reported to inhibit the growth of neuroblastoma and breast carcinoma cells, the intracluster redundancy has not been fully evaluated in gastrointestinal cancers. We tested a functional role of miR-17-5p in gastric cancer cells, given our patient data that miR-17-5p is significantly overexpressed in gastric cancer patients, compared with healthy volunteers. MiR-17-5p knock-down was performed in KATO-III human gastric cancer cell line, using commercially available lentiviral vectors targeting specifically miR-17-5p. Knock-down of miR-17-5p significantly decreased the proliferation rate of KATO-III cells. A smaller number of KATO-III colonies were formed in soft agar after miR-17-5p knock-down, suggesting a critical role of miR-17-5p in anchorage-independent cell growth. On the other hand, there was no change in the sensitivity of KATO-III cells to either fluorouracil or cisplatin after miR-17-5p knock-down, consistent with our patient data showing no association of miR-17-5p expression with chemotherapy response. These data suggests the non-redundant oncogenic function of miR-17-5p in vitro, leading to increased growth, but not decreased apoptosis of gastric cancer cells. Thus, inhibiting miR-17-5p is a promising therapeutic strategy against gastric cancer.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C145.
Abstract C148: LNA (locked nucleic acid)-based RNA antagonists mediate target mRNA silencing without transfection by simple addition to cell culture media
Authors: Zhang Yixian , Qu Zhengxing , Kim Steven , Shi Victoria , Liao Baisong , Kraft Patricia , Bandaru Raj , Sapra Puja , Greenberger Lee M. , Horak Ivan D.
Published: June 2012

Background: Since oligonucleotides do not penetrate cells well, most RNA-based therapies, such as antisense molecules and siRNAs use transfection conditions in tissue culture and delivery technologies in animals to facilitate entry of oligonucleotides into target cells. However, such vehicles are known to substantially alter cellular function and thereby significantly confound analyses. We have been developing LNA-based RNA antisense molecules to key cancer targets including survivin, HIF-1α, androgen receptor, GLI2, β-catenin, HER3, p110 alpha, and HSP27. LNA-based oligonucleotides were choosen since these molecules are highly stabile in plasma, have extraordinary high affinity to mRNA, and potently silence mRNA in cells under transfection conditions (IC50 in the high picomolar - low nanomolar range). We now report that LNA based RNA antagonist, when used in the high nanomolar - low micromolar range readily down modulate mRNA and protein of interests in multiple cell lines without any delivery means.

Material and Methods: In vitro, the ability of the antagonists to down modulate mRNA and cell growth was evaluated by qRT-PCR and MTS respectively. Cellular localization of the antagonists was evaluated by examining a FAM-labeled HER3 antagonist EZN-3920 under fluorescent microscope. In vivo, HER3 mRNA down-modulation and protein levels in tumors, which were grown on the flank of nude mice, were evaluated after intravenous administration of the antagonists.

Results: The down-modulation of mRNA in tissue culture-treated cells was quick, robust, long lasting, and specific followed by potent protein inhibition. Importantly, we show that mechanisms underlying the down-modulation correlated with nuclear localization of the LNA-ASO, suggesting that RNas H was the primary mechanism involved in target inhibition. Using this simple and effective approach, we were able to demonstrate that HER3, GLI2, and β-catenin were likely crucial growth and survival factors for certain cancer cells.

Conclusions: LNA-based RNA antagonists down-modulate target mRNA and protein in the absence of any agent to facilitate cell penetration. Such antagonists represent an attractive option for rapid, simple, and specific determination of gene function, target validation, and identification of drug-sensitive tumors for further in vivo evaluation. Beyond this, these results helps validate the use of intravenously administered LNA oligonucleotides simply prepared in saline, as is being done in on-going clinical trials with LNA-based RNA antagonists to HIF-1α and survivin.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C148.
Abstract C15: A novel panel of serum biomarkers distinguishes asthma from non-small cell lung cancer
Authors: Izbicka Elzbieta , Streeper Robert , Diaz Armando , Campos David , Long Thomas , Baek Sung , Mussman Rhys
Published: June 2012

Purpose: Asthma affects an increasing proportion of the population and may predispose sufferers to lung cancer. Frequent misdiagnosis of asthma and lack of early detection tests for lung cancer might be mitigated by better diagnostic tests. We used multiplexed immunoassays and mass spectrometry to identify serum biomarkers capable of distinguishing asthma from non-small cell lung cancer, the most common type of lung cancer.

Methods: Sera from normal subjects (NO; n = 300), patients with asthma (AST; n=180), and non-small cell lung cancer (LC; n = 334) were acquired from commercial vendors. Levels of 58 cytokines, chemokines and growth factors were quantified using multiplexed immunoassays. For biomarker discovery, specimens were digested with trypsin and analyzed by liquid chromatography electrospray ionization MS (LC ESI MS/MS). Proteins were identified using Mascot search software. Validation of select biomarkers identified by MS was achieved by immunodetection of target proteins in serum specimens. Data were reduced using inter-pathology comparisons with statistical significance determined using Student's t-test.

Results: Multiplexed immunoassays identified 29 analytes with significant (t<0.05) inter-pathology differences; 14 of these were highly significant (t < 0.0001). We fund a subset of biomarkers with unexpectedly large inter-gender differences and distinct gender-specific expression patterns. Serum levels of interleukin-13 and small inducible cytokine b11 (I-TAC) showed the greatest difference between AST, LC, and NO sera. MS analysis to date identified 11 differentially expressed proteins that included 3 putative yet unnamed proteins identified by gene sequencing and 1 protein product corresponding to chromosome X open reading frame 38. The presence of select proteins discovered by MS (syntaxin 11, cAMP-specific 3′,5′-cyclic phosphodiesterase type 7, and arginase) was confirmed and quantified by immunobloting.

Conclusion: We have identified a group of serum biomarkers having high inter-pathology discrimination power that are capable of differentiating AST and LC from normal controls and that may potentially be used in diagnostic tests for early detection of lung pathologies. Our experimental strategy is widely applicable to discovery and validation of biomarkers for diverse human diseases, response to a therapy, or pre-selection of patients for clinical trials.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C15.
Abstract C152: Therapeutic potential of histone deacetylase inhibitors in human liposarcomas
Authors: Mühlenberg Thomas , Pedeutour Florence , Fletcher Jonathan A. , Schuler Martin , Bauer Sebastian
Published: June 2012

Introduction: Systemic treatment of dedifferentiated liposarcomas (LPS) is limited to classical cytotoxic drugs, such as doxorubicin, ifosfamid or trabectedin. Recently, HSP90 inhibitors have shown evidence for clinical activity in patients with LPS. Histonedeacetylase inhibitors (HDACI) have been shown to disrupt hsp90 chaperone function with a favourable toxicity profile. Most LPS exhibit MDM2 amplification, resulting in p53 inhibition as important oncogenic event. We therefore sought to evaluate the therapeutic potential of HDACi alone and in combination with MDM2/p53 modulating drugs.

Materials and Methods: HDACi SAHA and LBH589 were evaluated in 4 liposarcoma cell lines (T449, T778, LPS141 and SW872). Biological consequences of HDAC inhibition alone and in combination with MDM2 inhibitors (nutlin-3 and RITA) were determined by cell viability assays, immunoblotting for p53- -related signalling pathways, effects on cell cycle and apoptosis as measured by flow cytometry and luminescence-based caspase activation assays.

Results: HDACi treatment (3 days) resulted in IC50 (cell viability) between 4µM and 8µM for SAHA and 25nM and 110nM for LBH589. Nutlin-3 showed strong antiproliferative effects in T449, T778 and LPS141 (IC50: 2–3µM), but not in SW872 (>10µM). Interestingly, SW872 exhibited a high sensitivity to RITA (IC50: 140nM) while all other liposarcoma cell lines were less sensitive (IC50 RITA: >10µM). Combinations of HDACI and Mdm2 inhibitors showed minor additive effects in cell viability assays.

Notably, HDACi treatment (SAHA 5µM, LBH589 50nM) resulted in substantial (80–90%) downregulation of p53 as measured by western blotting (after 24h) in all cell lines accompanied by induction of p21 (6- to 9-fold), and caspase 3 cleavage (9- 10-fold).

Nutlin-3 mediated induction of p53, p21 and Mdm2 was reduced when combined with SAHA or LBH589, while induction of apoptosis was strongly enhanced (40-fold) compared to baseline levels. Reactivation of p53 was less pronounced with RITA and no induction of p21 was observed.

Combination of nutlin-3 with SAHA or LBH589 resulted in a 2–3-fold increase of apoptotic cells (50–60%) compared to treatment with HDACi (10–30%) or nutlin-3 (32%) (alone). RITA exhibited only minor induction of apoptosis (6%) but showed similar additive effects when combined with HDACI (SAHA: 54%) and (LBH589: 40% apoptotic cells).

Conclusion: HDACi show antiproliferative and proapoptotic effects in liposarcoma cell lines. Notably, HDACi result in p53 inactivation. Combinations of HDACi with MDM2 inhibitors may greatly enhance the proapoptotic effects at doses that seem therapeutically achievable. RITA and nutlin-3 may exhibit differential activity in liposarcoma cell lines.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C152.
Abstract C158: IKKβ inhibition modulates NF-κB signaling and decreases aggressiveness of ovarian cancer
Authors: Hernandez Lidia , Hsu Sarah , Davidson Ben , Birrer Michael J. , Kohn Elise C. , Annunziata Christina M.
Published: June 2012

The NF-κB family of transcription factors has been implicated in the propagation of ovarian cancer, but the significance of constitutive NF-κB signaling in ovarian cancer is unknown. We hypothesized that constitutive NF-κB signaling defines a subset of ovarian cancer susceptible to therapeutic targeting of this pathway. To this end, we investigated the biological relevance of NF-κB in ovarian cancer cell lines using a small molecule inhibitor of IKKβ, and confirmed with RNA interference using stably expressed short hairpin RNA molecules towards IKKβ. We demonstrate here that the expression of IKKβ protein itself and the ovarian cancer-specific signature of IKKβ-regulated genes are related to poor outcome in independently collected sets of primary ovarian cancers. IKKβ signaling in ovarian cancer regulates the transcription of genes involved in a wide range of cellular effects known to increase the aggressive nature of the cells. We functionally validated the effect of IKKβ signaling on proliferation, invasion and adhesion in ovarian cancer cell lines. IKKβ was involved in all of these cellular functions, reflecting its modulation of the target genes identified. The diversity of functions controlled by IKKβ in ovarian cancer suggest that therapy targeted to this pathway could be efficacious if specific IKKβ inhibitor therapy is focused to patients whose tumors express a molecular profile suggestive of dependence on IKKβ activity.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C158.
Abstract C16: Prospective real-time analysis of p-cadherin expression to select patients into a phase I oncology trial
Authors: Davis Darren W. , Liu Wen , Jilani Iman , Zhang Yujian , Barlow John W. , Miller Louise M. , Winstanley Reba , Desai Jayesh , Alpaugh Kathy R. , Cooper Harry , Cohen Roger B. , Bang Yung-Jue , Oh Do-Youn , Kim Mi-Yeong , Bae Yoon-Ju , Countouriotis Athena , Shalinsky David R. , Kim Woo Ho
Published: June 2012

Effectively personalizing medicine depends upon selecting patients (pts) amenable to targeted therapies. We report the development of a custom immunohistochemistry (IHC) assay to identify P-cadherin positive expression in the archival biopsy of pts with solid malignancies. Validated reagents including a mAB to P-cadherin were incorporated into a custom IHC kit standardized across three Phase I sites (Seoul National University, Royal Melbourne Hospital, and Fox Chase Cancer Center).

Pts with a P-cadherin positive archival biopsy are being enrolled into an ongoing trial. Clinical results will be reported separately. P-cadherin positivity is defined by a scoring index (SI) [product of the Tumor Area Positive for P-cad x Staining Intensity]. Based on the literature, a SI ≥ 4 is associated with poor clinical outcome and was therefore required for pt entry into the trial. The IHC protocol was standardized across sites using positive and negative controls run in a masked fashion at each site and cross-examined and repeated by a central lab that had final decision-making authority (ApoCell, Inc., Houston, TX). Each site ran independent IHC assays and analyses by board-certified pathologists, and provided stained and unstained archival biopsy slides for independent, masked analysis by ApoCell. The turn-around time for central IHC analysis was ≤ 48 h.

Forty-four of 67 pts (65.7%) were found to be P-cadherin positive and eligible for entry into the trial. The distribution of enrolled patients by site is as follows: Australia (SI ≥ 4, n = 20), Korea (SI ≥ 4, n = 15), and USA (SI ≥ 4, n = 9). Median and mean SI values for the P-cadherin SI were 6 and 5.3 ± 2.9 (SD), respectively, with a range of 0 – 9. Clinical sites produced a SI equal to ApoCell in 56 of 67 pts (85.6% concordance rate). Among solid malignancies tested, a majority of gastric, colon and rectal cancers have demonstrated P-cadherin SI values ≥ 4. Successful execution of this IHC study demonstrates the feasibility of prospectively selecting pts based on IHC expression of P-cadherin using a custom standardized scoring index analysis across multiple clinical sites.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C16.
Abstract C160: Hepatocyte growth factor-MET receptor axis as a potential target for blocking cervical carcinoma metastasis into bones and bone marrow
Authors: Miekus Katarzyna , Kijowski Jacek , Drabik Grazyna , Majka Marcin
Published: June 2012

Introduction: Cervical carcinoma (CC) is one of the major causes of death among women suffering from tumor. The highest mortality is observed in the group of patients with late diagnosis and a metastatic disease. HGF/MET axis has been shown to be an important modulator of tumor cells motility and invasion. Recently we have shown that HGF/MET axis synergizes with SDF-1/CXCR4 axis in stimulating CC cell chemotaxis and activation of various intracellular pathways.

Objectives: In this study we focused on the role of MET receptor in CC.

Materials and Methods: CC cell lines (HeLa, HTB34 and HTB35) were transduced with MET shRNA and LacZ shRNA expressing virus and subsequently selected with blasticidin. To check receptors expression real-time PCR and flow cytometry was used. Migration was checked by chemotaxis and invasion assays. The level of various protein phosphorylation was estimated by western blot. NOD-SCID mice were used in in vivo experiments.

Results: Morphological assessment of MET negative cell lines revealed that one of them, HTB 35, has changed the morphology and cells became on average 2 times smaller than their control counterparts. The cells were also characterized by different proliferation rate when cultured under stressed conditions. Low oxygen (2%) or low serum condition caused strong > 50% inhibition of growth rate. Moreover, the migration and transmigration (through extracellular matrix proteins) toward HGF of all three cell lines with downregulated of MET was strongly inhibited. CC cells with downregulation of MET receptor showed inhibition of activation of several intracellular signaling pathways including RAS-MAPK, JAK-STAT and PI3K-AKT. We also performed in vivo study using NOD-SCID mice. All three cell lines were able to growth in these mice, however to different degree. HeLa and HTB 35 formed large tumors whereas HTB 34 formed small tumors. Growth of HeLa and HTB 34 cells was not inhibited by blocking MET receptor expression. However, HTB 35 tumor cells that lacked MET expression formed only very small tumors or no tumors at all. Cells present in these tumors were much more differentiated and matured. Moreover they formed structures similar to osteocytes and chondrocytes.

Summary: For the first time we shown here that HGF-MET axis activation significantly influences metastatic behavior of cervical carcinoma in vitro and in vivo and inhibition of this axis could be used as the new therapeutic strategy to block cervical carcinoma metastasis into bones and bone marrow.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C160.
Abstract C162: MET oncogen inhibition as a potentitial target for therapy of uveal melanoma
Authors: Abdel-Rahman Mohamed H. , Boru Getachew , Massengill James B. , Davidorf Frederick H.
Published: June 2012

Purpose: Uveal melanomas (UM), including choroidal, iris and ciliary body melanomas, are the most common intraocular malignancy in adult. The tumor spreads through a hematogenous route mostly to the liver in about 50% of the patients. Once metastatic, UMs are highly fatal and currently there is no available therapy for these tumors. Identifying novel therapy is crucial to improve the survival of these patients. The aim of this study is to investigate the utility of MET inhibition as a potential therapy for UM.

Methods: The frequency of MET activation (phospho-MET expression) in UM was studied using immunohistochemistry in 46 tumors. The half maximum inhibitory concentration (IC50) of selective MET inhibitor SU11274 was assessed on the five UM cell lines using cell proliferation assay. Inhibition of MET activation in UM cells treated with the SU11274 was confirmed using Western blot analysis. Effect of MET inhibition on cell migration was also studied.

Results: Our results showed that the majority (82.5%) of the 46 UM studied showed strong to moderate expression of phospho-MET. Selective MET blocking showed inhibition of tumor cell proliferation at an IC50 ranging from 2∼ 10 µM. In addition significant inhibition UM cell line migration was observed.

Conclusions: Our results indicate that MET is activated in a significant number of uveal melanomas. Our results also indicate the potential utility of MET inhibition as a target for therapy of UM.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C162.
Abstract C166: Cancer cells expressing TMPRSS4 colocalized with carbonic anhydrase IX (CAIX)-positive cells in lung and pancreatic carcinomas
Authors: Nguyen Tri-Hung , Havari Evis , Connors Timothy , Bagley Rebecca G. , McLaren Rajashree , Weber William , Nambiar Prashant R. , Madden Stephen L. , Teicher Beverly A. , Roberts Bruce , Shankara Srinivas
Published: June 2012

TMPRSS4, a cell surface transmembrane serine protease, is over expressed at the transcriptional level in pancreatic, colorectal and thyroid cancers compared with normal tissues. Recent studies have indicated a role for TMPRSS4 in tumor cell migration and tumor metastasis. We examined TMPRSS4 expression in dissected non-small cell lung cancer (NSCLC) tissues by quantitative RT-PCR and immunohistochemisty. At the transcriptional level, TMPRSS4 expression was elevated in adeno and squamous cell (SC) carcinomas when compared to the matching normal tissues (p=0.0035). At the protein level, over 100 tumor and normal specimens were examined with rabbit polyclonal anti-TMPRSS4 antibodies via immunohistochemistry. Adeno and SC carcinomas were positive for TMPRSS4 (p<0.05), while little or no staining observed on the normal tissue sections. TMPRSS4 protein expression in tumor samples correlated with mRNA qRT-PCR results. When similar experiments were performed on lung cancer cell lines, six out of 16 lines expressed TMPRSS4 mRNA, particularly NCI-H358 and NCI-H596 cells which exhibited high TMPRSS4 message with an average of 6000 and 5000 copies, respectively, relative to the 18S rRNA; however, no TMPRSS4 protein was detected either by Western blot or flow cytometry. When NCI-H358 cells were implanted into nude mice, TMPRSS4 was detected in the xenograft tumors via immunohistochemistry. When lung SC carcinoma and pancreatic adenocarcinoma were stained for TMPRSS4 and for the hypoxia marker, CAIX, TMPRSS4 positive cells were found to be adjacent to CAIX positive cells; however, no co-staining in the same cell was observed, suggesting that TMPRSS4 has a significant function with the adjacent hypoxic cancer cells in tumor tissues, and hypoxia may induce TMPRSS4 expression in xenograft tumors of NCI-H358 in which protein is not detectable under normal cell culture environment.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C166.
Abstract C172: Efficacy of AMG 479, alone and in combination with cisplatin, in ovarian carcinoma xenograft models
Authors: Beltran Pedro J. , Mitchell Petia , Cajulis Elaina , Moody Gordon , Chung Young-Ah , Wang Jon , Belmontes Brian , Radinsky Robert , Calzone Frank J.
Published: June 2012

AMG 479, a fully human monoclonal antibody raised to the type I insulin-like growth factor receptor (IGF1R), is currently being studied in the treatment of patients with ovarian carcinoma. We have previously shown that AMG 479 inhibits the growth of pancreatic and sarcoma tumor models by inhibiting IGF1R signaling through ligand blockade and receptor internalization and degradation. In the present study, we tested the ability of AMG 479 to inhibit the growth of four human ovarian carcinoma xenografts (OV-90, SK-OV3, TOV-21G, and OV-CAR3) in nude mice. Recent evidence has shown that multiple players in the IGF-axis are differentially regulated in human ovarian serous carcinomas, suggesting that the IGF1R pathway may play an important role in the growth of these tumors. The OV-90 model showed exquisite sensitivity to single-agent AMG 479, reaching significant (p<0.0001) tumor growth inhibition (TGI) of more than 90% with 100 or 300 g AMG 479/dose administered twice per week for the duration of the experiment. The OV-CAR3 model was also sensitive to single-agent AMG 479 and showed significant (p<0.0001) TGI of about 50% with 300 g AMG 479/dose, twice per week. The TOV-21G and SK-OV3 models showed resistance to AMG 479 with no significant TGI achieved at any dose. The OV-90 (sensitive) and TOV-21G (resistant) models were used to further characterize the AMG 479 response in a sensitive and a resistant model. AMG 479 effectively inhibited both IGF1R and Akt activity in the OV-90 model but not in the TOV-21G model in vitro. Furthermore, analysis of tumor xenografts collected at the end of the experiment revealed that TGI in the OV-90 model correlated with 50% receptor downregulation (total IGF1R), 90% inhibition of receptor activation (phospho IGF1R), and 50% inhibition of Akt activity, while no measurable inhibition in these analytes were detected in the TOV-21G xenografts. The potential benefit of combining AMG 479 with platinum-based therapy was assessed by combination with cisplatin in the moderately sensitive OVCAR-3 model. Treatment with AMG 479 (300 g/dose, twice per week) in combination with cisplatin (2.5 mg/kg, twice per week) resulted in significant (p=0.0001 vs. AMG 479, p=0.0005 vs. cisplatin) TGI compared with either therapy alone, suggesting that the two agents can be combined clinically for the treatment of ovarian carcinoma patients. Together, the data suggest that AMG 479 has therapeutic potential in treating ovarian carcinoma patients both as monotherapy and in combination with standard of care agents.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C172.
Abstract C174: Therapeutic reactivation of p53 through dual targeting of HDM2 and HDMX with a stapled p53 peptide
Authors: Bernal Federico , Wade Mark , Wahl Geoffrey M. , Walensky Loren D.
Published: June 2012

p53 is a transcription factor that induces cell cycle arrest or apoptosis in response to DNA damage and cellular stress, and thereby plays a critical role in protecting cells from malignant transformation. The E3 ubiquitin ligase HDM2 controls p53 levels through a direct binding interaction that neutralizes the transactivation activity of p53 and targets it for degradation via the ubiquitylation-proteasomal pathway. Whereas the HDM2-homologue HDMX lacks ubiquitin ligase function, it participates in regulating the p53 axis by heterodimerizing with HDM2 to form an active E3 ligase and/or by binding to p53 to prevent it from functioning as a transcriptional regulator. Loss of p53 activity, either by deletion, mutation, or HDM2/HDMX overexpression, is the most common defect in human cancer. Tumors expressing wild type p53 are rendered vulnerable by pharmacologic approaches that stabilize and upregulate p53. In this context, HDM2 and HDMX have emerged as independent therapeutic targets for restoring p53 activity and resensitizing cancer cells to apoptosis in vitro and in vivo. The small molecule nutlin-3 is an effective antagonist of the p53-HDM2 interaction. However, several studies have demonstrated the inability of nutlin-3 to disrupt the p53-HDMX complex, rendering tumor cells that overexpress HDMX nutlin-3-resistant. We have previously described the synthesis and characterization of a hydrocarbon-stapled α-helical p53 peptide (SAH-p53-8) that binds HDM2 with low nanomolar affinity, targets HDM2 in situ, and reactivates the p53 tumor suppressor pathway in HDM2overexpressing osteosarcoma cells. We now report that SAH-p53-8 binds HDMX with even greater affinity, co-immunoprecipitates with endogenous HDMX, and induces p53-dependent apoptosis in nutlin-resistant cancer cells that overexpress HDMX. Thus, by inserting a chemical staple into a peptide fragment of the p53 transactivation domain, we have generated the first bifunctional HDM2/HDMX inhibitor to investigate and modulate both targets in cancer cells. We further report a novel mechanism to maximize pharmacological activity through combined use of HDM2-specific and bifunctional HDM2-HDMX antagonists.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C174.
Abstract C18: Assessment of Gli1 expression during skin regeneration in mouse models and normal healthy volunteers
Authors: Wang Xi-De , Rowley Bruce , Li Hewei , Lang Lixin , Zhan Ping , Lewin Anne , Hawken Donald , Ho Ching-Ping , Huang Fei , Fargnoli Joseph , Alhers Christoph , Alland Leila , Van Tornout Jan M. , Bennett Kelly
Published: June 2012

Background: Inhibition of the Hedgehog pathway using Smoothened antagonists is emerging as a promising targeted therapy for cancers with pathway activating mutations or dysregulated expression. In support of the clinical development of BMS-833923 (XL139), an orally available, potent Smoothened antagonist, a pharmacodynamic assay was developed to measure Gli1 protein expression during the wound healing process.

Results: Gli1 protein and mRNA expression were first examined by immunohistochemistry or RT-PCR in mouse skin at baseline and in regenerating skin at various time points following the initial punch biopsy. The effects of BMS-833923 treatment on pathway expression profiles were then determined. The results showed that in control mice, Gli1 protein levels were elevated at as early as 3h and sustained for up to 72h. The intensities of Gli1 staining in both hair follicles and epidermis were greatly enhanced. However, in the mice treated with BMS-833923 at the time of initial biopsy, the magnitude and duration of Gli1 up-regulation were greatly reduced. While the Gli1 protein level was elevated within 6h, it returned to nearbaseline levels at 24h and remained low thereafter. Gli1 mRNA expression data support these observations. To better define the timing of potential Hedgehog pathway activation in human skin, an experimental medicine study was conducted to examine the expression of Gli1 after skin wounding and during regeneration in eighteen normal healthy volunteers. The baseline skin was sampled at the nape of the neck and the regenerating skin sample completely encompassed the initial wound. The intensities of both nuclear and cytoplasmic staining, as well as the percent cell staining positivity were scored. The data indicated that Gli1 protein was significantly up-regulated at 6h and 24h following wounding.

Conclusion: Gli1 protein level at 24h post-skin wounding assessed by immunohistochemistry is currently being used as a surrogate tissue pharmacodynamic measurement in a first-in-human multiple ascending dose trial in subjects with advanced or metastatic solid tumors.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C18.
Abstract C189: Active compound isolated from human urine induced differentiation and apoptosis in K562 chronic myelogenous leukemia cells and enhanced the effects of imatinib
Authors: Lai Gi-Ming , Yao Chih-Jung , Yeh Chi-Tai , Chen Suz-Wen , Yan Jiann-Long , Li Chi-Han , Chuang Shuang-En
Published: June 2012

Leukemia is a serious disease that affects both children and adults. Although all-trans retinoic acid (ATRA) and targeted drug such as imatinib could effectively treat acute promyelocytic (APL) and chronic myelogenous leukemia (CML), respectively, rare therapeutics is available when drug resistance or recurrence occurred. Searching for alternative agent for refractory patient is urgently needed. Our previous studies had found that a human urine extract CDA-2 could induce differentiation of HL60 APL cells as well as potentiate the effects of ATRA and imatinib on HL60 and K562 CML cells, respectively. Purification of active compound possessing these anti-leukemic effects from CDA-2 was thus performed. Recently, an active compound named as P18.9 was isolated. It induced differentiation of K562 CML cells as evidenced by the nitroblue-tetrazolium (NBT) reduction test. At higher dose, flow cytometry analysis showed that P18.9 induced sub-G1 apoptotic fraction of K562 cells and enhanced the sub-G1 fraction induced by imatinib at a scale of about two folds. Although P18.9 did not induce differentiation of HL60 APL cells, it substantially enhanced the ATRA-induced differentiation. This is the first study to isolate an active compound from human urine, which exerts anti-leukemic effects alone or cooperatively with clinical used drugs. The chemical structure of P18.9 had been identified and further subsequent structure modification is ongoing. Continuing the effort to develop P18.9 and its derivatives as novel therapeutics for refractory APL or CML patients is warranted.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C189.
Abstract C190: BMS-777607, a potent small molecule c-MET inhibitor, inhibits prostate cancer progression in vitro
Authors: Dai Yao , Siemann Dietmar
Published: June 2012

Overexpression of c-MET is highly associated with prostate cancer progression and therapy resistance. BMS-777607 is a selective and potent c-MET inhibitor that was advanced to clinical evaluation. Herein we report the effects of BMS-777607 in androgen-independent prostate cancer cells (PC-3, DU145). When assessed using an MTT assay, BMS-777607 treatment was found to inhibit cell growth in the absence of hepatocyte growth factor (HGF), with an IC50 >5.0 uM for both cell lines. In a wound-healing assay, BMS-777607 suppressed PC-3 and HGF-induced DU145 cell motility in a dose-dependent fashion (IC50 values of 3.0 and 0.5 uM, respectively). Moreover, BMS-777607 efficiently blocked HGF-triggered DU145 cell scattering in a cell scatter assay; with 40% inhibition at 0.2 uM. Using a Matrigel-based Transwell assay, BMS-777607 also inhibited PC-3 cell invasion in a dose-dependent manner, with 60% inhibition occurring at 1 uM. Western blot analysis demonstrated that BMS-777607 blocked c-MET autophosphorylation, and downregulated multiple downstream kinases involved in c-MET signaling pathway, including Src, Fak and Akt. These data indicate that BMS-777607 inhibited prostate cancer progression in vitro including proliferation, motility, scattering and invasion. Furthermore, the finding of more potent effects on cell migration and invasion than cell proliferation suggests that this small molecule c-MET inhibitor could have utility as an anti-metastatic agent for the treatment of advanced prostate cancer.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C190.