bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract A26: Aromatase over-expression results in the development of ductal growth abnormalities in the mammary gland of a conditional transgenic mouse model
Authors: Diaz-Cruz Edgar S. , Nakles Rebecca E. , Sugimoto Yasuro , Brueggemeier Robert W. , Furth Priscilla A.
Published: June 2012

Estrogens are required for the development of the normal mammary gland; however, studies suggest that estrogen and its metabolites may have mutagenic and carcinogenic potential in the mammary gland. Aromatase is key to estrogen biosynthesis, but its over-expression may play a role in the development of breast cancer by increasing mitotic activity in breast epithelial cells. Therefore, it is highly valuable to have mouse models with increased local estrogen production to use to develop optimal therapeutic and chemopreventive strategies to treat these lesions. The purpose of this study was to evaluate if increased local estrogen production led to the development of mammary gland preneoplasia. To test our hypothesis we developed a conditional transgenic mouse model to control the spatial and temporal aromatase expression in the mammary gland. Mammary glands were collected at 4 and 12 months of age for morphological, histological, gene and protein expression studies in both MMTV-rtTA/tet-op-Aromatase and wild-type mice. RT-PCR analysis of aromatase mRNA confirmed that the gene was conditionally expressed in the mammary gland as compared to control mice. Aromatase activity assays confirmed a significantly higher enzyme activity in the mammary glands of MMTV-rtTA/tet-op-Aromatase compared to wild-type. Mammary glands were evaluated for morphological changes using confocal microscopy, whole-mount and hematoxylin and eosin staining. Aromatase over-expression resulted in a higher incidence of morphological and histological abnormalities such as hyperplastic alveolar nodules, ductal dysplasia, ductal carcinoma in situ, and the unexpected persistence of terminal end buds at 12 months of age. These changes were associated with ERK1/2 and AKT activation. In summary, we generated a novel transgenic mouse system to investigate the role of aromatase over-expression in mammary epithelial cells during mammary gland development. Increased aromatase activity resulted in the development of ductal growth abnormalities and increased ductal density. This model represents a valuable tool to investigate the effect of estrogen over-production in the mammary gland microenvironment at specific times during development.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A26.
Abstract A261: In vitro characterization of AMG 900, an orally active small molecule inhibitor of aurora kinases in phase 1 clinical trials
Authors: Chung Grace , Kendall Richard , Geuns-Meyer Stephanie , Payton Marc
Published: June 2012

Background: The aurora family of serine-threonine kinases (Aurora-A, -B, -C) regulate cell-cycle progression in mammalian cells. Aurora kinases A and B are essential for proper chromosome congression, segregation, and cytokinesis during mitosis, whereas aurora kinase C function appears restricted to male meiosis. Aurora kinase B is responsible for the direct phosphorylation of histone H3 on serine-10 (p-histone H3). Aurora kinase A and B expression is elevated in a variety of human cancers and is associated with advanced clinical staging and poor prognosis. The emergence of aurora kinases as key mitotic regulators and their potential role in tumorigenesis has focused substantial interest in developing selective small molecule inhibitors for the treatment of human cancers.

Objective: The aim of this study was to characterize the in vitro effects of AMG 900 in tumor cell lines, including cell lines resistant to paclitaxel and three well-characterized aurora kinase inhibitors.

Results: AMG 900 is a novel ATP competitive small molecule inhibitor that is highly potent and selective for aurora kinases A, B, and C. In cells, AMG 900 inhibits autophosphorylation of aurora kinases A and B as well as the phosphorylation of histone H3. The predominant cellular response of tumor cells to AMG 900 treatment is aborted cell division without a mitotic arrest, which leads to endoreduplication and cell death. The effect of AMG 900 was tested in a panel of 26 tumor cell lines to evaluate its potential to inhibit cell proliferation across multiple tumor types. AMG 900 inhibited the proliferation of tumor cell lines at low nanomolar concentrations (EC50 values 1–6 nM). Importantly, AMG 900 was equally potent against multidrugresistant (MDR) cell lines expressing ATP-binding cassette transporters (P-gp and BCRP1), whereas paclitaxel and three aurora kinase inhibitors (AZD1152, MK-0457, and PHA-739358) showed a loss of potency in these MDR-positive cell lines compared with matched parental cell lines. To further investigate potential mechanisms of resistance, an HCT116 cell line was adapted to grow in the presence of AZD1152, a selective inhibitor of aurora kinase B. The resulting AZD1152-resistant HCT116 subline harbors a mutation in one allele of the aurora kinase B gene (W221L substitution) and was negative for P-gp and BCRP1 expression. AMG 900 induced an accumulation of cells with ≥4N DNA content (EC50 value 5 nM) and inhibited colony formation in this AZD1152-resistant HCT116 subline.

Conclusion: To our knowledge, AMG 900 is the first described inhibitor of aurora kinases A, B, and C that has uniform in vitro potency in MDR-positive tumor cell lines. AMG 900 is currently undergoing phase 1 clinical evaluation in patients with advanced solid tumors.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A261.
Abstract A262: Antiangiogenic proprieties of E-3810, a new dual inhibitor of VEGF and FGF receptors
Authors: Giavazzi Raffaella , Oliva Paolo , Taraboletti Giulia , Berndt Alexander , Colombo Sonia , Valbusa Giovanni , Cavalletti Ennio , Cereda Roberta , Damia Giovanna , Camboni Gabriella
Published: June 2012

E-3810, 6-[[7-[(1-aminocyclopropyl)methoxy]-6-methoxy-4-quinolyl]oxy]-N-methyl-naphthalene-1-carboxamide, is a novel small molecule that potently and selectively inhibits VEGFR1-3 and FGFR-1 tyrosine kinases. E-3810 has shown antitumor activity against various types of cancer xenografts with good tolerability. The anti-angiogenic proprieties of E-3810 were investigated in vitro on endothelial cells and in vivo in angiogenesis-induced models and human tumor xenografts.

E-3810 inhibited VEGF- and FGF-2-induced HUVEC (human umbilical vein endothelial cells) proliferation with an IC50 of 40–50 nM. The ability of E-3810 to inhibit angiogenesis in vivo was assessed in the Matrigel plug assay, where angiogenesis is induced by FGF-2 embedded in Matrigel pellets, implanted subcutaneously in mice. Treatment with E-3810 (20 mg/kg p.o., daily for 6 days) completely inhibited the angiogenic response measured by hemoglobin content.

The effect on tumor angiogenesis was investigated in the human A498 renal carcinoma xenografted subcutaneously in nude mice. Oral treatment with E-3810 for up 30 days at the optimal antitumor dose of 20 mg/kg led to significant tumor growth delay (Treated -Controls = 24 days). E-3810 significantly decreased blood vessel density and increased vessel maturation index (assessed by immunostaining with anti CD31 and CD31/ SMA antibodies); this was accompanied by decreased expression of stroma collagen IV and increased tumor necrosis.

A498 xenografts growing in nude mice were also analyzed by DCE-MRI (dynamic contrast-enhanced magnetic resonance imaging) before and after 5 and 12 days of oral treatments with 20 mg/kg E- 3810. The kinetic analysis of the distribution of the contrast agent (Gadocoletic Acid Trisodium Salt, B22956/1, Bracco S.p.a., Italy) showed significant reduction in perfusion, calculated applying the IAUC method over the first 30 minutes post contrast injection, in E-3810 treated tumors compared with both baseline values and untreated controls already after 5 days of treatment. These results show a significant antiangiogenic activity of E-3810, which is likely to contribute to its potent antitumor activity.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A262.
Abstract A29: Development of liposomal curcumin as a new potential anticancer agent
Authors: Smith Judith A. , Mathew Lata , Mach Claire M. , Santiago Katherine , Helson Larry
Published: June 2012

Previously, pre-clinical dose finding tolerance study in a human xenograft pancreatic cancer model of intravenous liposomal curcumin determined 20 mg/kg TIW was to be the optimum dose and schedule for anticancer activity. Follow up pharmacokinetic and dose finding studies in a health rat model evaluated doses up to 40 mg/kg was not associated with weight loss, hematological, serologic, or dose-limiting toxicity. In canine model, a single-dose finding tolerance study ranging from 2 mg/kg up to 40 mg/kg revealed the maximum tolerated dose (MTD) of liposomal curcumin to be 20 mg/kg or 540 uMol/L of curcumin maximal blood concentration. Toxicity observed at this dose level was characterized by a brief single episode of reversible hematuria. The dose limiting toxicity was observed at a single dose of 40 mg/kg of liposomal curcumin. Following dose on day 1, life threatening toxicity followed within 48 hours with the dogs exhibiting irreversible acute hemolysis with hematuria, over 60% blood loss, and associated serologic abnormalities. A control cohort of dogs infused with the same quantity of liposome contained in the 40/mg/kg dose was without ensuing toxicity. These changes suggest the mechanism of hemolysis following 40mg/kg curcumin is due to an oxidant effect. Following acute hemolysis, the iron chelation activity of curcumin could contribute to unremitting anemia by blocking iron reutilization. In conclusion, at concentrations below the canine MTD of 20 mg/kg, curcumin acts as an anti-oxidant, and acts as a pro-oxidant at higher concentrations. The disparity between rodent and canine sensitivity to liposomal curcumin may be due to species differences in pharmacokinetics and/or curcumin metabolism. Ongoing study will define liposomal curcumin pharmacokinetic parameters at the MTD in canine mode as well define tolerability of multiple dosing in dogs. In addition hematological studies are being conducted to determine the mechanism of hemolysis that was observed at higher dose levels and identify potential biomarkers for predicting toxicity. Preliminary data suggests liposomal curcumin will have promising anticancer activity.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A29.
Abstract A30: Integrated analysis of genomic data and annotations in cancer research
Authors: Ustach Carolyn V. , Han Haiyong , Kim Seungchan , Hostetter Galen , Diep Caroline H. , Hidalgo Manuel , Barret Michael , Keats Johnathan J. , Von Hoff Daniel , Kingsley Christopher
Published: June 2012

Many cancer researchers are currently generating heterogeneous genomic data sets containing mRNA expression, array based comparative genomic hybridization (aCGH), and other information from multiple patients. In the near future, data sets will likely include proteomics, methylomics, and high throughput sequencing data. Analytical tools that integrate these different data types to prioritize genes for further study have lagged, however. With this in mind, we have implemented a standalone software application that integrates CGH and mRNA expression profiling data. This application implements several different methods to generate ranked lists of genes and genomic regions based on breakpoint frequency, aberration frequency, and the impact of copy number changes on mRNA expression. The software is Java GUI based, with abstracted object models that can be extended as new genomic data types and annotations become available. We used this application to analyze aCGH datasets generated from 22 primary pancreatic tumor xenografts and mRNA expression datasets from 16 of the 22 samples. Genes which were located near a putative breakpoint and whose expression levels were high in a majority of the xenografts samples were considered putative targets. Preliminary IHC analysis of pancreas primary tumor tissue microarrays confirms one of these putative targets, Chemokine (CXC motif) Ligand 5 (CXCL5). By allowing cancer researchers to correlate genomic aberrations with expression data, we believe that this application will generate more meaningful targets for further biomarker identification and/or drug target discovery.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A30.
Abstract A33: CuraBase: An integrated relational database and online collaboration platform for cancer drug development
Authors: Husler William , Nosrati Mehdi , Rothenberg Mace L. , Korn W. Michael
Published: June 2012

The extraordinary increase in the number of novel, targeted agents for the treatment of cancer has resulted in the generation of a vast amount of clinical and preclinical data at an ever-quickening pace. Currently, no standardized system exists allowing for systematic collection, organization, and analysis of such data. Curabase was developed to provide scientists involved with cancer drug development with quick access to relevant data on molecular targets, targeted agents, and clinical trial results. The system consists of a relational database capturing information on new drugs, drug targets and pathways, and clinical trial outcomes using a standardized format. Incorporated into this database is a pathword-protected, firewalled online collaboration platform allowing for secure information exchange within workgroups. Database content can be annotated and thus expanded based on individual needs. The relational database structure allows for generation of comprehensive and relevant data output. For example, tables summarizing clinical trial results from various drugs targeting particular molecular targets or pathways can be generated on demand. This relational database has the potential to enhance analysis, communication, and collaboration on drug development projects in academia and industry.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A33.
Abstract A42: Characterization of EGFR and leucine-rich repeats and immunoglobulin-like domain proteins (LRIG1–3) in esophageal carcinoma with emphasis on patient survival
Authors: Wu Xuping , Hedman Håkan , Bergqvist Michael , Bergström Stefan , Henriksson Roger , Gullbo Joachim , Lennartsson Johan , Hesselius Patrik , Ekman Simon
Published: June 2012

Background: Esophageal carcinoma is the seventh most common cause of cancer-related death in the Western world and the incidence is increasing. The signal transduction system of epidermal growth factor receptor (EGFR) is of major importance for tumor growth in several types of malignancies and EGFR-targeted therapy has been shown to able to regulate tumor growth. Leucine-rich repeats and immunoglobulin-like domain protein 1 (LRIG1) is an endogenous negative regulator of EGFR signaling. The functions of LRIG2 and −3 are at present not known, however, it has been postulated that they might also be involved in the regulation of growth factor signaling and tumor growth. In the present study, we investigated the clinical implications of the expression of EGFR and LRIG1–3 in esophageal carcinoma as well as the correlation between their expression levels and the chemosensitivity of esophageal carcinoma cell lines.

Patients and Methods: A total of 86 patients diagnosed with esophageal carcinoma were included in the study and their tumors were investigated for the expression of EGFR and LRIG proteins by immunohistochemistry. Further, a total of nine esophageal carcinoma cell lines were investigated for their expression of EGFR, LRIG1, −2 and −3 by quantitative RT-PCR and for their sensitivity to commonly used chemotherapeutics by a cytotoxicity assay.

Results: Survival analysis using Kaplan-Meir analysis showed that the staining intensities of EGFR and LRIG2 were significantly associated with short survival (p=0.025 and p=0.03, respectively), whereas the extent of staining of LRIG1 was associated with long survival (p= 0.045).

Chemosensitivity studies demonstrated a significant correlation between LRIG1 expression levels and sensitivity to cisplatin (r=−0.74), docetaxel (r=−0.69) and vinorelbine (r=−0.82) in the investigated esophageal carcinoma cell lines.

Conclusion: In the present study, the expression of EGFR and LRIG2 correlated negatively, whereas LRIG1 correlated positively, with survival of the esophageal carcinoma patients. Furthermore, a correlation between LRIG1 expression and chemosensitivity was observed, indicating a possible role in chemoresistance. The functions of LRIG1 and −2 and their connection with EGFR warrants further investigation.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A42.
Abstract A45: Upregulation of the small GTPase Rap1 and the insulin-like growth factor I receptor in breast epithelium occurs in carcinoma in situ
Authors: Furstenau Dana K. , Mitra Nandita , Wan Fei , Lewis Robert , Feldman Michael D. , Fraker Douglas L. , Guvakova Marina A.
Published: June 2012

With use of screening mammography, the diagnosis of breast carcinoma in situ (CIS) is increasing worldwide. Although classified as a noninvasive precancer, the presence of this lesion increases risk for developing invasive cancer. The molecular markers associated with this transition remain uncertain. Recent reports indicated that activation of the small GTPase Ras - related protein 1 (Rap1) in human breast cancer cells associated with cell invasion in vitro and tumorigenicity in nude mice (Itoh, M. et al.. Cancer Res, 67: 4759–4766, 2007). The objective of this study was to test the hypothesis that the alteration in the levels of Rap1 protein expression is associated with invasion of breast cancer cells in humans. Using an image-based uniplex method, we performed quantitative protein profiling on 90 surgical samples representing breast cancer progression series and including 17 histologically normal tissues, 16 benign lesions; 10 pure CIS, 5 CIS with co-existing invasion, and 42 invasive carcinomas. The expression of Rap1 was found increased in pure CIS and significantly increased in CIS with co-existing invasion (P=0.0010) and invasive carcinoma (P<0.0001) compared with noncancerous tissue. Furthermore, the expression of Rap1 positively correlated with the expression of the insulin-like growth factor type I receptor (IGF-IR) in invasive carcinomas (Spearman's = 0.39; P = 0.014; n = 42), but not in normal/ benign epithelia (= -0.39; P = 0.841; n = 33). Moreover, both Rap1 and IGF-IR significantly discriminated invasive from non-invasive tissues, with receiver operating characteristic (ROC) area under the curve, AUC=0.78 for Rap1, AUC= 0.82 for IGF-IR. Our studies identified measurable changes in Rap1 and IGF-IR protein expression during the transition of breast epithelium to CIS and invasive cancer. These findings warrant further investigation on IGF-IR-Rap1 as a potential dual biomarker of early alterations associated with breast epithelium transition from CIS to invasive breast cancer in humans.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A45.
Abstract A46: Pharmacodynamic biomarkers for OSI-906, an insulin-like growth factor-1 receptor (IGF-1R) tyrosine kinase inhibitor, in cancer patients with advanced solid tumors
Authors: Gedrich Richard , Poondru Srini , Stephens Andrew , Logan Tiffany , Hart Karen , Buck Elizabeth , Carden Craig , Lindsay Colin , Kaye Stanley , Kim Edward , Evans Jeff , Puzanov Igor , Epstein David , Miglarese Mark
Published: June 2012

Background: OSI-906 is a potent small molecule inhibitor of the insulin-like growth factor 1 receptor (IGF-1R), a receptor tyrosine kinase that is overexpressed in several human cancer types and implicated in resistance to chemotherapy. Disruption of the growth hormone-IGF1 signaling axis can affect levels of biomarkers such as IGF1, growth hormone (GH) and insulin and can affect glucose homeostasis, making these potentially useful markers for demonstrating the activity of IGF-1R inhibitors in solid tissues.

Methods: Two phase I dose escalation trials were initiated in cancer patients with advanced solid tumors who received OSI-906 on either continuous or intermittent dosing schedules. Pharmacodynamic (PD) and pharmacokinetic (PK) assessments were included as secondary objectives of these studies. Circulating biomarkers, including total IGF1, IGFBP3 and non-fasting GH and insulin levels, were measured at various times during the dosing cycle (21 days for continuous dosing; 14 days for intermittent dosing) and changes were correlated with plasma concentrations of OSI-906.

Results: To date, patients have been treated with up to 450 mg QD and 200 mg BID on the continuous dosing schedule and up to 750 mg QD on the intermittent dosing schedule. Maximum tolerated dose (MTD) was determined to be 400 mg QD and 150 mg BID for continuous dosing and 600 mg QD for intermittent dosing. Preliminary analysis of the PK data suggests that the exposure of OSI-906 increased with dose. Median plasma concentrations of OSI-906 at MTD exceeded the predicted concentration required for efficacy based on the preclinical models (1 µM). Total IGF1 concentrations in plasma were increased relative to predose levels at doses ≥ 450 mg QD on the intermittent dosing schedule and ≥ 150 mg QD or 40 mg BID on the continuous dosing schedule. Elevations in non-fasting plasma insulin levels were also observed in patients with plasma concentrations of OSI-906 exceeding 5000 ng/mL. PK/PD relationships were observed for IGF1 and insulin. OSI-906 did not affect plasma IGFBP3 levels in most patients. Analysis of additional biomarkers, including GH, is ongoing.

Conclusions: At or below MTD, plasma concentrations of OSI-906 could be achieved that exceed concentrations required for anti-tumor efficacy based on the preclinical models. The pharmacodynamic data indicate that pharmacologically-relevant concentrations of OSI-906 were achieved in tissues involved in regulating the GH-IGF1 signaling axis. Together, these PK and PD data indicate that the current recommended dosing regimens may provide sufficient exposure for activity against solid tumors.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A46.
Abstract A47: Somatic mutations in human cancer cell lines predict sorafenib response
Authors: Rhodes Daniel R. , Kaufmann Scott H. , Meng Xue Wei , Wyngaard Peter , Leopold Judith S.
Published: June 2012

It is now widely accepted that a spectrum of somatic alterations occur in human cancer and that particular alterations are associated with response or lack of response to anti-cancer therapies. Here, we undertook a systematic correlation analysis of somatic mutations, copy number aberrations and drug response across 300+ human cancer cell lines. We studied the RAF and VEGFR inhibitor, sorafenib, evaluating associations between sorafenib response and somatic mutations in 35 common cancer genes and high-level amplifications and homozygous deletions in more than 100 minimal common regions of gain and loss, which include many known oncogenes and tumor suppressor genes. Notably, we found that NRAS mutations, among other alterations, were significantly associated with sensitivity to sorafenib (odds ratio = 7, p-value = 0.003), whereas activating BRAF mutations were strongly associated with a lack of response (odds ratio = 4.9, p-value = 0.012). Specifically, 64% (7 of 11) of NRAS mutant cell lines were sensitive to sorafenib, while only 20% (34 of 170) of NRAS wild-type lines were sensitive. On the contrary, just 6.7% (2 of 30) of BRAF mutant lines were sensitive to sorafenib. Because NRAS mutations occur frequently in leukemia and no leukemia cell lines were included in the original analysis, we selected 5 NRAS mutant and 5 NRAS wild-type leukemia cell lines for further evaluation. Consistent with our previous observation, the four most sensitive leukemia cell lines were all NRAS mutant, each line exhibiting greater than 50% growth inhibition after 72 hours of exposure to 4uM sorafenib. These results confirm the original association and suggest that across both solid tumors and hematologic malignancies, NRAS mutations may identify a subset of cancers that are likely to respond sorafenib. In addition, this work demonstrates that systematic analysis of somatic alterations and drug response across a large collection of cancer cell lines is capable of discovering novel predictive biomarkers.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A47.
Abstract A56: Chemically induced alkaline phosphatase on osteosarcoma stem cells derived from a novel osteosarcoma cell line as a potential molecular target
Authors: Saini Vaibhav , Shoemaker Robert H.
Published: June 2012

Osteosarcoma is the sixth most common pediatric tumor in the under 15 age group. Relapse observed after chemotherapy is associated with < 20 % survival. Osteosarcoma stem cells (OSCs) have been implicated in the poor response to chemotherapy and ultimate relapse of osteosarcoma. Thus, to achieve successful osteosarcoma therapy, it may be crucial to target OSCs. To this end, a molecular target that is overexpressed in osteosarcoma cells and OSCs and either not expressed or underexpressed in normal cells is desirable. While the search for such a targeting molecule is ongoing, we endeavored to test the utility of a chemical treatment to induce overexpression of a potential molecular target in osteosarcoma cells and OSCs. We hypothesized that a chemical cocktail could be used to induce overexpression of bone specific alkaline phosphatase (bsALP) in both osteosarcoma cells and OSCs derived from a novel osteosarcoma cell line (CHA59). Towards this goal, an OSC-enriched culture was derived from CHA59 using serum-free culture medium. This was accomplished by changing the cell growth media, as recently reported for other cell lines, from 10% fetal bovine serum-containing medium to serum-free medium containing 15% KnockOut Serum Replacement (serum substitute (SS)). This resulted in a rapid change in cell growth from adherent monolayers (in serum) to floating spheres (in SS). Monolayers, containing osteosarcoma cells and a few OSCs, and OSC-enriched spheres were then treated with a chemical cocktail (0.1 µM dexamethasone, 50 M ascorbic acid, and 10 mM beta-glycerolphosphate) that has been previously reported to induce the expression of bsALP in mesenchymal stem cells. A colon tumor cell line, HT-29, served as the negative control for ALP induction. Nine days post-treatment, ALP expression was monitored by Fast Red staining. Cocktail treated monolayer and OSC-enriched spheres stained intensely for ALP compared with the untreated control and the HT-29 (cocktail treated) negative control. To conclude, the results support our hypothesis that ALP can be overexpressed in both monolayer and OSC-enriched spheres. This opens the possibility of targeting therapeutic drugs, gene therapy vectors, and novel nanotechnology to OSCs along with the osteosarcoma cells using anti-bsALP monoclonal antibodies, which is our future goal.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A56.
Abstract A57: The telomere targeting agent sodium metaarsenite (KML001) exhibits activity in taxane-resistant prostate cancer (PC) cells
Authors: Burger Angelika M. , Zhang Bin , Nolan Patrick , Brucz Bradley , Hussain Arif
Published: June 2012

Background: Tumor growth is sustained by cancer stem cells (CSC), a rare fraction of cells expressing high levels of drug efflux pumps responsible for therapy failure and tumor recurrence. The enzyme telomerase is critical to maintaining the self-renewal properties of CSC. We have recently found that sodium metaarsenite (KML001), a drug in phase I/II clinical trials in Europe and the US, can target both the catalytic subunit of telomerase and telomeres. PC cell lines appear to be among the most sensitive tumor cell types to KML001. In this study, we examined chemotherapy-naïve and taxane (paclitaxcel/Pac and docetaxel/Doc)-resistant DU145 PC cell lines for stem cell content, and the potential for KML001 to inhibit their mature and stem cell subpopulations.

Methods: Methyltetrazolium (MTT)-based assays were used to determine the IC50 and IC100 values, and the relative resistance of DU145, DU145/Doc50 and DU145/Pac200 cells to KML001, docetaxel and paclitaxel. Fluorescence activated cell sorting (FACS) was employed to determine the prostate stem cell surface marker CD44, the side population and Pgp content in these cells. Telomerase activity was evaluated by a PCR ELISA assay and telomere length was assessed by Southern blots. Cells were treated with IC50 and IC100 concentrations of KML001 for 72 hrs to investigate its effects on the side population.

Results: We found that while DU145/Doc50 and DU145/Pac200 cells were 2,000- and 400-fold resistant to docetaxel and paclitaxel, respectively, they retained sensitivity to KML001 (IC50 = 4µM). Resistance to taxanes was associated with expression of Pgp; parental DU145 cells expressed Pgp in 0.24% of the cell population, DU145/Doc50 in 47% and DU145/Pac200 in 79% of the cells. Moreover, Pgp expression correlated with the detection of a side population (SP), a cell fraction known to be enriched for CSC. The SP was 3.7% in DU145 cells and 56%–67% in the taxane-resistant cells. In contrast, telomerase activity and telomere lengths remained unchanged in all three cell lines. Exposure of the various DU145 cell lines to KML001 for 72 hrs resulted in a dramatic reduction in the SP. Similar to the Pgp inhibitor verapamil, KML001 reduced SP cells to ∼93% of controls in both parental and taxane-resistant cell lines. However, unlike verapamil, KML001 was able to reduce the mature cell population to the same extent as the SP.

Conclusion: Overall, our data in the DU145 model demonstrate that KML001 can eradicate both mature drug-resistant and prostate cancer stem cells, which may offer a new treatment approach in this disease.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A57.
Abstract A74: Activation of EGFR by proteasome inhibition requires HB-EGF in pancreatic cancer cells
Authors: Sloss Callum M. , Wang Fang , Palladino Michael , Cusack James C.
Published: June 2012

Resistance to drug treatments underlies the high lethality of pancreatic ductal adenocarcinoma (PDA). We have previously shown that the EGFR/HER4 ligand HB-EGF plays an important role in chemoresistance of pancreatic cancer cell lines to various chemotherapeutics. Additionally we and others have recently identified that proteasome inhibition is a promising therapeutic option in this highly refractory disease. The pleiotropic effects of proteasome inhibition include the activation of anti-apoptotic signaling pathways as well as the expected pro-apoptotic signals. These anti-apoptotic pathways include increased heat shock protein expression, cFLIP induction and activation of EGFR, AKT and the MAP kinases all of which can reduce the apoptotic potential of this class of drug.

In the current study, we sought to determine the mechanism behind the activation of EGFR in response to proteasome inhibition in pancreatic cancer cells. We found that the second generation proteasome inhibitor NPI-0052 induced EGF, HB-EGF and epiregulin mRNA expression. Using both a specific inhibitor of HB-EGF activity (crm-197) and lentiviral mediated shRNA knock down of ligand expression, we discovered that HB-EGF but not EGF or epiregulin is required for activation of EGFR in response to proteasome inhibition. Additionally we found that a broad spectrum inhibitor of MMP/ADAM activity (GM6001) was able to prevent proteasome inhibitor induced activation of EGFR. Taken together these results indicate that HB-EGF expression and cleavage is required for EGFR activation in response to proteasome inhibition. This suggests that HB-EGF plays a prominent role in pancreatic cancer chemoresistance to proteasome inhibition and that targeting both HB-EGF and the proteasome may make an effective combination treatment for this highly refractory disease.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A74.
Abstract A77: Hydrostatic pressure sensitizes TRAIL-mediated apoptosis through activating mitochondrial pathway in H460, lung carcinoma cell
Authors: Oh Sangnam , Lee Eunil , Kwon Daeho
Published: June 2012

TRAIL (tumor necrosis factor related apoptosis inducing ligand) is a member of the TNF superfamily and one of the promising anticancer drugs with high specificity for cancer cells. Accumulating preclinical studies demonstrate that the TRAIL can effectively induce the apoptosis of tumor or transformed cells, but not normal cells; hence, promoting the development of TRAIL-based cancer therapy. Recent studies for strategy of enhancement of the TRAIL-mediated apoptosis have been investigated for improving typical cancer therapy by combining treatment with other chemo- or radiation and small molecules.

Environmental stimuli such as physiological or mechanical conditions can influence the proliferation, differentiation, apoptosis and premature senescence through activating critical signaling pathways with the dependent on the kinds of stimuli or cell types like as chemicals. However, until present, there are a few reports to postulate molecular mechanisms of cancer therapy combined with environmental stimuli. Here, using new culture system, compressed air into two atmospheres (hydrostatic pressure, HP), we explore the molecular mechanism of HP stimulation coupled with TRAIL-mediated apoptosis in lung carcinoma cell H460 (one of non small cell lung carcinoma cells). Interestingly, HP treatment combined with TRAIL significantly induced the apoptosis by 2.0-fold in H460 cells compared to TRAIL only treated cells.

We show the HP-potentiating apoptosis of H460 is characterized by activation of intrinsic pathway which defined as caspase 8-mediated Bid cleavage and subsequent the disruption of mitochondrial membrane potential and cytochrome c releases. This was accompanied by the increased ROS generation and down-regulated inhibitor of apoptosis (IAPs) such as XIAP and survivin. HP-enhanced apoptosis is completely abrogated by Z-IETD-FMK, caspase8 inhibitor. Furthermore about 37% increased expression of DR5 was observed in cells treated with TRAIL and HP. Although the HP-induced sensitization of apoptosis also shows in another lung cancer cells and cells from different organs, human normal cells remains resistant without any damages from the treatment coupled with HP.

Taken together, these findings suggest that HP as one of the mechanical and physiological stimuli, can act as an adjuvant tool for attractive cancer treatment mediated by TRAIL. Our ongoing studies are evaluating the specific mechanism of anticancer activity via TRAIL and HP using transcriptome and proteome tools and controlling initiation of apoptosis signaling in TRAIL-based cancer therapy.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A77.
Abstract A80: Metronomic cyclophosphamide induces macrophage recruitment in regressing human and rat gliosarcoma xenografts
Authors: Doloff Joshua C. , Waxman David J.
Published: June 2012

Metronomic chemotherapy has shown improved therapeutic activity over conventional schedules, both in preclinical studies and in initial clinical trials for several malignancies. Metronomic cyclophosphamide treatment (140 mg/kg BW every 6 days) induces dramatic, near-complete regression of established xenografts of human U251 and rat 9L gliosarcoma in s.c. scid mouse models, whereas maximum tolerated dose cyclophosphamide effects moderate tumor growth delay without sustained regression. Metronomic cyclophosphamide-induced regression of 9L tumors is accompanied by a reduction in microvessel density, suppression of CD31 expression, induction of the anti-angiogenic factor thrombospondin-1 (TSP1), and impaired drug uptake. These findings are consistent with the high intrinsic sensitivity of proliferating endothelial cells to metronomic chemotherapy shown by others, and with the prevailing view that anti-angiogenesis is a major factor in the therapeutic effectiveness of metronomic chemotherapy. However, VEGF receptor inhibition induces substantially greater anti-angiogenesis in these tumor models, yet does not induce major tumor regression. Presently, we investigate whether non-anti-angiogenic mechanisms may contribute to the observed regression of U251 and 9L gliosarcomas, and demonstrate metronomic cyclophosphamide-induced host macrophage recruitment. This recruitment is associated with induction of the mouse (host) factors PEDF (pigment epithelium-derived factor; Serpinf1) and PAI-1 (plasminogen activation inhibitor-1; Serpine1), with the levels of PEDF RNA being strongly correlated (r=0.89) with TSP1 RNA in large sets of 9L and U251 tumors isolated various times after initiation of metronomic cyclophosphamide therapy. Notably, PAI-1 and TSP1 are both expressed by tumor-associated macrophages. Metronomic cyclophosphamide also induced Fas, which stimulates macrophage recruitment during inflammation, and the macrophage marker CD68, supporting the involvement of tumor-associated macrophages in the response to metronomic chemotherapy. These findings highlight the ability of metronomic chemotherapy to induce macrophage recruitment, thereby activating innate immunity leading to regression of established gliosarcomas and potentially other tumor types. Supported in part by NIH grant CA49248 (to DJW).

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A80.
Abstract A82: HHER2-positive breast cancer cells resistant to trastuzumab and lapatinib lose reliance upon HER2 and become sensitive to the multitargeted kinase inhibitor sorafenib
Authors: Capellero Sonia , Valabrega Giorgio , Cavalloni Giuliana , Zaccarello Gianluca , Petrelli Annalisa , Migliardi Giorgia , Milani Andrea , Neia Caterina Peraldo , Gammaitoni Loretta , Favero Francesco , Moggio Aldo , Giordano Silvia , Aglietta Massimo , Montemurro Filippo
Published: June 2012

HER2 targeting with trastuzumab has changed the prognosis of breast cancer patients carrying amplification and/or overexpression of this oncogene. Despite this progress, however, resistance to trastuzumab occurs in the vast majority of patients. Newer anti-HER2 therapies, like the dual tyrosine-kinase inhibitor (TKI) lapatinib, show antitumor activity in a limited proportion of patients, indicating that HER2 can be still exploited as a target after trastuzumab failure. However, due to the high proportion of patients that fail to respond to these alternative strategies, it is reasonable to assume that cells escaping HER2 targeting may rely on alternative pathways not involving HER2 to sustain their growth. Their knowledge is relevant for exploiting new therapies. To investigate this hypothesis we generated a human HER2 overexpressing breast cancer cell line resistant to trastuzumab and lapatinib (T100) and we have characterized it genetically and molecularly.

In T100 cells, the previously proposed mechanisms of resistance to HER2 inhibitors did not explain cell escape. Notably, silencing HER2 by shRNA did not affect the growth of T100 cells, suggesting loss of reliance upon HER2. Among the alternative pathways that we explored, altered levels of the antiapoptoptic proteins Mcl-1 and Survivin were observed. This suggested the possibility of targeting resistant cells with the multitarget inhibitor sorafenib. Moreover, sorafenib, nearly inactive in parental cells, strongly inhibited the in vitro growth of T100 cells.

In conclusion, we provide a new model where the activation of HER2 independent mechanisms sustains the growth of tumor cells, significantly increasing the sensitivity to sorafenib.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A82.
Abstract A83: Liposomal doxorubicin induces Golgi-dependent apoptosis in hormone-refractory prostate cancer cell lines (HRPC)
Authors: Fabbri Francesco , Carloni Silvia , Brigliadori Giovanni , Ulivi Paola , Tesei Anna , Montanari Marco , Amadori Dino , Silvestrini Rosella , Zoli Wainer
Published: June 2012

Purpose: The current efficacy of HRPC therapy is disappointing and new agents and therapeutic modalities are urgently required. The aims of the present work were to examine the mechanisms of action and the in vitro activity of liposomal doxorubicin in HRPC cell lines.

Methods: Doxorubicin (Doxo) and liposomal Doxo (Myocet®) activity was assessed by SRB test, and apoptosis by TdT assay, in DU145 and DU145-R HRPC cell lines. Intracellular drug incorporation and localization was analyzed by fluorescence image microscopy, CD95 and GD3 expression by cytofluorimetry, and protein marker alterations by Western blot.

Results: Myocet® showed a higher cytotoxic activity than Doxo in both cell lines, particularly in docetaxel-resistant DU145-R cells after a 72-h exposure, with 70% of apoptotic cells at 1/10 of the plasma peak concentration. In addition, cytofluorimetry and fluorescence microscopy revealed that Myocet® achieved higher intracellular concentrations than Doxo. The liposomal anthracycline formulation was found to concentrate primarily in the Golgi apparatus and induced a significant increase in the expression of CD95 death receptor, GD3 ganglioside, and caspase-2 and -3 in both cell lines. Interestingly, Myocet® also induced a stronger Mcl-1 downregulation than Doxo, although this was more pronounced in the parental DU145 cell line.

Conclusions: Myocet® demonstrates a remarkable activity in HRPC cells, particularly in those previously resistant to Doxo, probably due to its high intracellular drug concentration and Golgi localization. Myocet® appears to have two mechanisms of action: a conventional anthracycline-induced DNA damage-dependent apoptosis, and a Golgi-dependent cell death pathway, confirmed by the increase in CD95, GD3 and caspase-2 and -3 expression. Our results strengthen the hypotheses that the Golgi apparatus may act as a stress sensor, and that organelles other than mitochondria may trigger cell death, further proof that combinatorial targeting of diverse cell death pathways may improve the efficacy of anti-cancer strategies. In conclusion, our data advocate further evaluations of Myocet® as a second line treatment for advanced HRPC.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A83.
Abstract A85: Discovery of highly potent and selective B-Raf Kinase inhibitors
Authors: Wang Xiaolun , Berger Dan M. , Salaski Edward J. , Torres Nancy , Hu Yongbo , Jeremy Levin I. , Powell Dennis , Wojciechowicz Donald , Collins Karen , Frommer Eileen
Published: June 2012

B-Raf kinase, a serine/threonine protein kinase, is a component of RAS-Raf-MEK-ERK signaling pathway and plays a central role in cell growth and survival. B-Raf mutations have been found in about 8% of all cancers with the highest incidence in melanomas (66%). Thus a potent Raf inhibitor could have a significant impact in treating cancers that are dependent on this pathway for survival and proliferation signaling. As part of our program to prepare potent and structurally novel B-Raf kinase inhibitors, we designed a series of substituted pyrazolo[1,5- ]pyrimidines. Molecular modeling studies were utilized to validate the preparation of this series, as well as to optimize analogs to ultimately provide B-Raf inhibitors with subnanomolar IC50s. The modeling result also suggested these compounds bind to the active conformation of B-Raf. These pyrazolo[1,5- ]pyrimidine derivatives have moderate selectivity for B-Raf vs. C-Raf. Selected examples were screened for inhibition of a panel of 24 kinases and found to be highly selective. In addition to their enzymatic potency, the cell antiproliferative activities of these inhibitor were evaluated in B-Raf mutant cell lines (A375,WM266-4) and a cell line with wild type B-Raf (Caco-2). Selective inhibition of the B-Raf mutant cell lines with IC50 values less than 10 nM was observed for the most potent compounds. Finally, protein immunoblot analyses confirmed the inhibition of this MAPK signaling pathway by these analogs.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A85.
Abstract A86: Design, synthesis, and SAR of focal adhesion kinase (FAK) inhibitors
Authors: Roberts Walter Gregory , Berliner Martin , Coleman Kevin , Emerson Erling , Griffor Matt , Hulford Catherine , Jani Jitesh , Kath John , LaGreca Susan , Lin Jing , Lorenzen Marianne J. , Marr Eric , Martinez-Alsina Luis , Patel Nandini , Richter Daniel , Roberts Erika , Autry Christopher , Ung Ethan , Felix Vajdos , Vetelino Beth , Wessel Matthew , Whalen Pamela , Xu Huiping , Yao Lili
Published: June 2012

Focal adhesion kinase (FAK) is a non-tyrosine kinase that localizes to focal adhesion plaques. It is activated in response to intergin binding to cellular ligands and when phosphorylated inhibits anoikis allowing for anchorage independent cell growth. Recent studies have shown increased FAK expression and phosphorylation status in many types of invasive and aggressive human tumors strongly suggesting FAK is a possible target for anticancer chemotherapy. Literature, in house HTS and de novo studies identified 2, 4-diaminopyrimidines as potent FAK inhibitors. Early SAR efforts quickly determined that smaller substituents, particularly CF3, were optimal in the C5 position. Parallel medicinal chemistry strategies were executed for the C2 and C4 positions. These studies suggested that substituted aryl and fused heteroaryl groups at the C2 position in conjunction with substituted phenyl and heterocycles at the C4 position imparted optimum activity and metabolic stability. Inhibitor-FAK co-crystal structures were utilized to guide in the SAR strategy around the 2, 4-diaminopyrimidine template which afforded several lead compounds. The team's effort culminated in the advancement of PF-562,271 as a potent and reversible inhibitor of FAK (kinase IC50 of 2 nM and cell IC50 of 5 nM) that is > 100x selective against a long list of non-target kinases. In summary, detailed SAR studies were executed on the 2, 4-diaminopyrimidine templates that produced potent inhibitors of FAK with improved ADME properties, and identified a novel and potent series of FAK inhibitors that are selective against most other kinases and have shown activity in clinical trials. This poster will present design, synthesis, challenging chemistry, optimization, and complete inhibitor chemical structures of lead analogs.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A86.
Abstract A92: Chemoprevention of small cell lung carcinomas in mice by the retinoid X receptor agonist bexarotene
Authors: Wang Yian , Wen Weidong , Yi Yijun , Zhang Zhongqiu , Lubet Ronald A. , You Ming
Published: June 2012

In the present study, we examined the effect of bexarotene (Targretin) and budesonide in chemoprevention of small cell lung carcinoma (mSCLC) using a lung specific knockout model of Rb1 and p53. Upon treatment with bexarotene, tumor incidence, number, and load were significantly reduced (p < 0.05). Budesonide treatment trended to inhibition, but the effect was not statistically significant (p > 0.05). Immunohistochemical staining indicated that bexarotene treatment decreased cell proliferation and increased apoptosis in tumors. The Rb1/p53 gene targeted mouse appears to be a valuable model for chemopreventive studies on human small cell lung cancer (hSCLC). Our results indicate that the retinoid X receptor (RXR) agonist bexarotene may be a potent chemopreventive agent in this cancer type.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A92.