bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract C114: The KidsCancerKinome: Validation of CDK2 as potential drug target in pediatric tumors
Authors: Molenaar Jan J. , Westerhout Ellen M. , Boer den Monique L. , Clifford Steve C. , Delattre Olivier , Geoerger Birgit , Lanvers Claudia , Pietsch Torsten , Serra Massimo , Shipley Janet , Vassal Gilles , Versteeg Rogier , Verschuur Arnauld C. , Caron Huib N.
Published: June 2012

The KCK consortium has validated CDK2 as potential drug target in six highly malignant pediatric tumor types (i.e Ewing sarcoma, osteosarcoma, rhabdomyosarcoma, neuroblastoma, medulloblastoma and ALL). The stepwise procedure started with the selection of genes that can be targeted using small molecule inhibitors. Drug target genes and signal transduction routes were studied using Affymetrix mRNA profiles of over 500 tumors and cell lines. CDK2 and cell cycle signal transduction genes showed expression patterns that were significantly different from reference tissues and expression was related to poor prognosis. The next step to evaluate the potential drug targets consisted of targeted knock down using RNA interference. Inhibition of CDK2 resulted in significant phenotypes in several pediatric tumor models. Cell lines from most tumor types showed growth inhibition and differentiation. Neuroblastoma cell lines showed extensive apoptosis. Highly interesting was the synthetic lethal relation between CDK2 and the driving oncogene MYCN. These findings warranted further evaluation using the CDK2 inhibiting small molecule Roscovitine. This compound was tested in the core panel of pediatric tumor cell lines and more extensive in tumor types that showed high sensitivity for the compound. Neuroblastoma cell lines were most sensitive with IC50 in the low uM range. Neuroblastoma, which showed high sensitivity for Roscovitine, are now being evaluated in in vivo mouse xenograft models.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C114.
Abstract C130: Plucked human scalp hairs: Potential utility as a surrogate tissue for the assessment of therapies targeting the androgen receptor
Authors: Growcott Jim , Brady Ged , Pickering Karen , Walker Jill , Lovick Susan , Chetty Raj , Kevill Helen , Prahladan Manoj , Grimes Emma , Harrison Elliott , Mefo Timothy , Hodgson Darren
Published: June 2012

Objective: Evaluate whether plucked human scalp or eyebrow hairs obtained from male elderly healthy normal volunteers (HNVs) are suitable for assessing RNA expression patterns linked to the androgen receptor.

Procedure: For each of 12 HNV subjects plucked eyebrow and scalp hairs were obtained at 2 time points. For each time point up to three individual anagen hairs were selected for RNA extraction and representative cDNA amplification. For each sample RNA quality was assessed by agarose gel electrophoresis and cDNA quality assessed by qPCR analysis of 3 reference or “housekeeping” genes. Expression levels of a panel of 20 androgen receptor regulated genes were measured by qPCR for samples passing both RNA and cDNA quality criteria.

Results : Of the 72 scalp hairs analysed 64 (89 %) passed both RNA and cDNA quality criteria with 23 out of 24 (96%) sample collection points yielding 2 or more analysable hairs. This high success translated into available data for both time points in 11 out of 12 HNVs (92 %). In contrast, for the 72 eyebrow hairs analysed only 2 (3 %) passed RNA quality criteria with none of the collection points yielded 2 or more analysable hairs. Of the 20 test genes qPCR analysis identified a short list of 7 genes with expression levels similar to “housekeeping” genes indicating that they should be reliably detected in the majority of scalp hairs. These results are in keeping with a previous HNV study of younger male and female donors and indicate that plucked scalp hairs may be of value in the early phases of clinical development of androgen receptor based therapies.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C130.
Abstract C143: Expression profiles of miRNA, FFPE, and nonamplified samples for diagnostic tests using a supersensitive microarray
Authors: Sudo Hiroko , Ueda Yoji , Kuroda Toshihiko , Takizawa Satoko , Nomura Osamu , Akiyama Hideo , Nobumasa Hitoshi
Published: June 2012

We have developed a novel gene expression microarray system, 3D-Gene™, featuring micro-columnar structure on the platform made of black resin and micro-bead agitation for efficient hybridization. With these technological characteristics, 3D-Gene™ achieves dramatic reduction of background noise, exceptionally high sensitivity and reproducibility, and high correlation with qRT-PCR methodology particularly for genes with low expression. In addition to the general high performance, however, the medical devices in clinical settings must be versatile for a variety of biomarker types as well as sample types. Employing this microarray, we further developed a highly sensitive system to detect attomole-level miRNA which is reported to be a novel biomarker for some cancers. Moreover, we were able to detect gene profiles reproducibly and accurately from formalin-fixed paraffin-embedded (FFPE) tissue which is known to be stored worldwide in a large number for retrospective studies. Finally, we developed a RNA sample preparation protocol with no amplification process, resulting in a quick and cost-effective protocol with high accuracy. Taken together, we developed systems for gene expression detection using a high performance microarray suitable for a wide range of clinical applications. 3D-Gene™ could be a powerful tool for drug target discovery and clinical diagnostic for various diseases.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C143.
Abstract C155: External Qi of Yan Xin Qigong induces apoptosis and inhibits migration and invasion of estrogen-independent breast cancer cells by down-regulating Akt/NF-κB signaling
Authors: Yan Xin , Shen Hua , Jiang Hongjian , Hu Dan , Zhang Chengsheng , Wang Jun , Wu Xinqi
Published: June 2012

Background: The concept External Qi of Qigong refers to the technology and ability of “Qi deployment” therapy and health preservation of traditional Chinese medicine (TCM). External Qi therapy of TCM has long been one of the medical practices in China. Long-term clinical observations and ongoing studies have shown positive effects of external Qi of Yan Xin Qigong (YXQ-EQ) on cancer patients. Recently, the molecular and cellular mechanisms underlying YXQ-EQ's antitumor effects have begun to be addressed. In the present study we investigated YXQ-EQ's effects on growth, migration, invasion and apoptosis of estrogen-independent breast cancer MDA-MB-231 cells and the underlying molecular mechanisms.

Methods: Cytotoxic effects were evaluated by trypan blue exclusion assay, MTS assay and/or clonogenic assay. Apoptosis was assessed by DNA fragmentation and cleavage of caspases and poly(ADP-ribose) polymerase. Quantitative analysis of apoptosis and cell cycle distribution was carried out by flow cytometry. Migration and invasion assay was performed in Transwell Boyden chambers and Matrigel-coated Boyden chambers, respectively. NF-κB activity was determined by electrophoresis mobility shift assay. Akt phosphorylation and NF-κB target gene expression was evaluated by Western blot analysis.

Results: YXQ-EQ treatment of MDA-MB-231 cells induced a time-dependent reduction in viability, clonogenic growth inhibition, and apoptosis. Furthermore, YXQ-EQ blocked migration and invasion of MDA-MB-231 cells. Biochemically, YXQ-EQ markedly inhibited constitutive and EGF-induced Akt phosphorylation. YXQ-EQ also substantially repressed NF-κB activity, resulting in decreased expression of antiapoptotic Bcl-2, Bcl-XL, XIAP and survivin proteins.

Conclusion: Our findings suggest that YXQ-EQ may induce apoptosis and inhibit migration and invasion of MDA-MB-231 cells through the repression of Akt/NF-κB signaling.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C155.
Abstract C171: On- and off-target effects of telomere uncapping G-quadruplex ligands based on pentacyclic acridinium salts
Authors: Stevens Malcolm FG , Hutchinson Ian , McCarroll Andrew J. , Hummersone Marc , Williams Huw EL
Published: June 2012

Antitumor properties of the potent G-quadruplex ligand 3,11-difluoro-6,8, 13-trimethyl-8H-quino[4,3,2-kl]acridinium methosulfate (RHPS4) are driven by telomere uncapping events. RHPS4 has good selectivity for quadruplex DNA over duplex DNA as measured by surface plasmon resonance (Kquad 1.1 × 107 M−1); Kdup 3.4 × 105 M−1). In preclinical models of solid tumor xenografts RHPS4 potentiates the activity of camptothecins but good response is dependent on the timing of the drug sequence employed. These experiments teach how an agent of this novel class might be used clinically in combination with chemotherapeutic agents. Although RHPS4 has several ‘drug-like’ qualities (synthetic accessibility, water solubility, cellular uptake), a preliminary toxicological study revealed that the compound is a potent hERG inhibitor (IC50 0.2 M) and has additional receptor interactions, notably with muscarinic M1, M2 and M3 receptors. In an anaesthetized guinea pig cardiac model RHPS4 increased QTcB interval at doses of 5 and 10 mg/kg. Modifications to the peripheral substituents around the pentacyclic core have identified agents with differing on- and off-target effects. Removal of the N(13)-methyl group of RHPS4 to generate an uncharged molecule drastically reduced affinity for the target quadruplex DNA coupled with reduced pharmacological liabilities; increasing the size of the onium group at N(13) from Me to Et reduced affinity for quadruplex DNA (Kquad 0.6 × 106 M−1) and increased hERG inhibition (IC50 0.04 M). After a significant redesign program two isomeric compounds have been identified, 2- and 3-acetylamino-8,13-dimethyl-8H-quino[4,3,2-kl]acridinium iodides with ‘win-win’ profiles - better selectivity for quadruplex DNA and markedly reduced hERG liability (for the 2-acetylamino compound Kquad 2.4 × 107 M−1; Kdup 3.8 × 105 M−1; hERG IC50 3.7 M): for the 3-acetylamino isomer Kquad 2.5 × 107 M−1; Kdup 4.4 × 105 M−1; hERG IC50 18 M). Molecular modeling studies on the favored 3-acetylamino derivative indicate that the pentacyclic core acts as a surrogate K+ stabilizing the G-quadruplex structure with the acetylamino side-chain projecting into a quadruplex groove.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C171.
Abstract C185: Head and neck squamous carcinoma cells are susceptible to the attenuated vesicular stomatitis virus expressing suicide genes
Authors: Porosnicu Mercedes , Liu Jingfang , Lyles Douglas E.
Published: June 2012

Background: The predominant loco-regional progression of Head and Neck Squamous Cell Carcinoma (HNSCC) makes it suitable for testing oncolytic viruses. Recent work from several laboratories including ours has focused on developing vesicular stomatitis virus (VSV) as a replication-competent anticancer viral vector. Attenuated strains, such as M protein mutant VSV (M51R) are more likely to be promoted in clinical research. The major shortcoming of viral therapy is related to the large proportion of tumor cells remaining uninfected. We are addressing this limitation in two ways: the expression of the cytosine deaminase-uracil phosphoribosyltransferase (C:U) suicide gene by the rVSV vector, expected to produce a strong bystander effect and the exploitation of the radiosensitizing potential of the 5-fluorouracil (5-FU) chemo-drug produced intratumorally, as well as of the VSV virus itself.

Methods: We determined the susceptibility to oncolytic VSV in a collection of HNSCC cell lines and in patient HN primary tumor cells by measuring: 1) the level of GFP expression after infection with rVSV-GFP and M51R-GFP; 2) the viral production by plaque assay and 3) cell death by Trypan blue exclusion. The extent of defectiveness in interferon pathways was tested by employing pre-treatment with IFN and INF-blocking antibodies before the infection with the oncolytic VSV. We have generated the rVSV-C:U and M51R-C:U viruses and we tested our collection of HNSCC cell models to infection with these viruses in the presence and absence of 5-fluorocytosine. SQ20B cell line is a model of radiation resistant HNSCC. Regular SQ20B and SQ20B infected with low-dose M51R-VSV were evaluated by clonogenic assay.

Results: 1) All HNSCC cell models tested are susceptible to the infection with the rVSV but many present a degree of resistance to the attenuated M51R-VSV. 2) Human Primary HNSCC cells are more resistant than the HNSCC cell lines to the M51R-VSV; 3) The activation of IFN pathways is contributing to the resistance to M51R-VSV. 4) Addition of the suicide gene C:U to the attenuated VSV model significantly increased the oncolytic effect in vitro; 5) HNSCC cells infected with M51R-VSV become more radiation sensitive.

Conclusions: The decreased oncolytic effect of the attenuated M51R-VSV can be overcome by the expression of suicide genes such as the yeast C:U fusion model. The demonstrated radiosensiting potential of the M51R-VSV virus as well as the known radiosensitizing effect of the generated chemotherapeutic 5-FU can be further exploited against the loco-regional HNSCC tumors. Finally, we have important new information, both from cell lines and patient tissues, for proof-of-principle that cancers can down-regulate antiviral responses.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C185.
Abstract C188: Nanoantibodies and other engineered antibody domains as candidate cancer and infectious disease therapeutics
Authors: Vu Bang K. , Gong Rui , Xiao Xiaodong , Chen Weizao , Dimotrov Dimiter S.
Published: June 2012

Engineered single antibody domains are small (sizes about 12–14 kDa i.e. about 10-fold smaller than the size of a typical full-size antibody) and can access targets and epitopes that are not accessible by larger molecules. We have constructed two large (size 2×1010) VH-based libraries of high diversity (Chen et al, JMB, 2008, and submitted) from which several high affinity domain antibodies against HIV and cancer-related proteins were identified and characterized with potential use as therapeutics (Chen et al, PNAS, 2008, and unpublished). We have also engineered human antibody constant domains (CH2) as scaffolds which are highly stable, soluble, and express at high levels (Rui et al, JBC, 2009, and unpublished). Based on these new scaffolds several libraries have been constructed, and binders were selected and characterized (Xiao et al, BBRC, 2009; Bang et al, unpublished). Further engineering to confer additional functions (nanoantibodies) is in progress.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C188.
Abstract C193: Biological evaluation of novel multi-targeted Aurora-B kinase inhibitor TAK-901 in xenograft models
Authors: Satou Takashi , Hoshino Takashi , Farrell Pamela , Zhang Lilly , Elliott Sarah , Fabrey Robyn , Lee Bumsup , Paraselli Bheema , Ishino Seigo , Nomura Toshiyuki , Teratani Mika , Ohta Yoshikazu , de Jong Ron
Published: June 2012

Aurora kinases play an essential role in cell division, specifically orchestrating several intricate steps during the mitotic phase of the cell cycle. The human genome encodes for 3 Aurora kinase genes, designated Aurora A, B, and C. Aurora A localizes to centrosomes and spindle poles and is required for mitotic spindle assembly, whereas Aurora B is a chromosome passenger protein required for phosphorylation of histone H3, chromosome segregation, and cytokinesis. Expression levels of Aurora A and B kinases are frequently elevated in human cancers making them attractive targets for therapeutic intervention. TAK-901 is an Aurora B kinase inhibitor with multi-targeted inhibitory activity against other kinases important in malignancy. In the present study, we investigated the pharmacodynamic effects and in vivo activity of TAK-901 in human tumor xenograftmodels. In solid tumor xenograft models, potent dose-dependent antitumor activity was observed against HCT116 (colon), H460 (large cell lung) and A2780 (ovary) cancer models by twice daily intravenous dosing on 2 consecutive days per week for 3 cycles. TAK-901 also displayed potent activity against several leukemia models such as subcutaneously implanted MV4-11 and HL60 (AML). In the A2780, HCT116, and HL60 models, TAK-901 induced tumor regression at higher dose levels, including complete regression of A2780 tumors at 30 and 45 mg/kg/injection. When dosed in combination with chemotherapeutic agents, such as irinotecan in the HCT116 model or daunorubicin in the HL60 model, TAK-901 produced additive effects. Following intravenous administration of 20 and 40 mg/kg TAK-901 in the A2780 xenograft model, histone H3 phosphorylation was completely suppressed for at least 6 hours and in the 40 mg/kg treated tumors did not return to control levels during the 12 hour time course. After multiple doses of TAK-901, polyploidy was observed in the tumor samples. TAK-901 drug levels remained constant throughout the time course in tumor tissues whereas plasma levels declined steadily. These in vivo biomarker studies demonstrate that TAK-901 induces pharmacodynamic responses consistent with Aurora B kinase inhibition, which correlates with effective retention of TAK 901 in tumor tissues. Taken together, these preclinical data emphasize the therapeutic potential of TAK- 901 in the treatment of diverse human malignancies. TAK-901 is currently under investigation in Phase I clinical trials.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C193.
Abstract C200: Effects of indirubin derivatives on the FLT3 activity and growth of acute myeloid leukemia cell lines
Authors: Choi Sang-Un , Ahn Jin Hee , Kim Min-Jin , Yoo Miyoun , Kim Eunbin , Kim Young-Kyoon , Ryu Shi Yong , Han Sun-Young
Published: June 2012

Indirubin is an active constituent of traditional Chinese recipe used for the treatment of chronic myelocytic leukemia. In this study, inhibitory activity of indirubin and its derivatives toward Fms-like tyrosine kinase 3 (FLT3) was examined. Indirubin-3′-oxime(IO) and 6-bromoindirubin-3′-oxime(BIO) showed potent inhibitory activity against FLT3 with 50% inhibitory concentration (IC50) of 79 nM and 254 nM, respectively. Meanwhile, indirubin and 6-bromoindirubin exhibited negligible effect on FLT3 inhibitory activity up to 10 µM. We also tested the cytotoxicity of those compounds against acute myeloid leukemia cell lines; MV4;11 cells harboring constitutively activated form of FLT3 and RS4;11 cells with wild type FLT3. IO and BIO potently inhibited the growth of MV4;11 cells with IC50 of 30 nM and 61 nM, respectively. On the other hand, RS4;11 cells were far less sensitive to those compounds with IC50 values of 3600 nM and 820 nM, respectively. In the cell cycle assay, IO arrested the cell cycle of MV4;11 cells at G1 phase, and increased dead cell population at sub-G1 phase about 25 and 50% at 0.1 and 1.0 µM after 48 hours, respectively. These results strongly suggest that the derivatives of IO have potentials to be developed for novel anti-leukemic agents.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C200.
Abstract C204: Inhibition of c-Met signaling in combination with cisplatin and 5-fluorouracil significantly enhances antitumor effects in gastric cancer models
Authors: Efferson Clay , Lee Linda , Tammam Jennifer , Ware Chris , Deshmukh Sujal , Harrington Elizabeth , Pan Bo-Sheng , Reilly John F. , Majumder Pradip K. , Marshall Christopher Gary
Published: June 2012

Gastric cancer is currently the second most frequent cancer-related cause of death with an incidence rate of approximately one million cases annually. The present worldwide standard of care treatment regimen for gastric cancer is the combination of 5-fluorouracil (5-FU) and platinum analog (cisplatin). Many receptor tyrosine kinases are deregulated in gastric cancer including erbB2, c-Met, and EGFR. The purpose of our study was to dissect the role of c-Met signaling in gastric cancer and test the effects of our c-Met inhibitor (MK-8033) alone and in combination with the standard of care agents 5-FU and cisplatin. As a monotherapy, MK-8033 potently inhibited proliferation in a panel of gastric cancer cell lines in vitro. When administered simultaneously with cisplatin and 5-FU, the effects of the combination were diverse, ranging from additive to antagonistic. Altering the treatment schedule by the addition of 5-FU and cisplatin prior to MK-8033 resulted in an overall combination benefit across the panel. In a gastric cancer xenograft model, while MK-8033 alone significantly inhibited tumor growth progression, the co-administration of 5-FU and cisplatin with MK-8033 showed greater benefit than either the monotherapy or the standard regimen. In vitro cell cycle and survival analysis support the observed in vivo data, and provides a mechanistic rationale for the observed combination benefit. Our data suggest the combination of c-Met inhibitors with standard of care agents 5-FU and cisplatin may be beneficial in gastric cancer patients with activated c-Met signaling.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C204.
Abstract C210: LIM kinase small molecule inhibitors block tumor cell invasion
Authors: Crighton Diane , Charles Mark , Trivier Elizabeth , Hooper Steven , Scott Rebecca , Riddick Michelle , Brookfield Joanna , Ekwuru Tennyson , Stockley Martin , Foxton Caroline , Williams Juliet , Raynham Tony , Sahai Erik , Olson Michael , Hammonds Tim
Published: June 2012

The invasive potential of carcinomas greatly contributes to their ability to metastasize, a process which is estimated to cause 90% of all human cancer deaths. The LIM kinase (LIMK) family of Ser/Thr kinases sit at a hub of signaling pathways downstream of the Rho family of GTPases. Functionally, LIMK is directly involved in regulating the activity of cofilin, a family of proteins which modulate cell movement through reorganization of the actin cytoskeleton network. LIMK is up-regulated in a number of invasive cancer cell lines and metastatic breast and prostate tumors, whilst an increase in LIMK activity has been shown to cause increased cellular invasion in multiple model systems.

We demonstrate that LIMK inhibition by siRNA reduces the invasive capacity of MDA-MB-231-Luc breast cancer cells in an in vitro matrigel invasion assay and reduces fibroblast lead collective invasion in a co-culture organotypic model. Potent, selective, small molecule LIMK inhibitors have been identified that inhibit LIMK in vitro with sub-nanomolar activity in biochemical assays and low micromolar activity in cells as measured by cofilin phosphorylation. Treatment of MDA-MB-231-Luc cells or tumor associated fibroblasts with these small molecule inhibitors decreased cell invasion in vitro with minimal cellular toxicity. Collectively, these data further validate LIMK as an anti-invasive therapeutic target in tumor cells and demonstrate the potential utility of LIMK inhibitors in metastatic disease.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C210.
Abstract C241: Fine needle aspiration biopsy as a new method for monitoring of tumor microenvironment after treatment with polymeric drugs
Authors: Betka Jaroslav
Published: June 2012

Introduction: Cancer therapy seems to be, according to the latest findings, a combination of direct tumor cytotoxicity and the reaction of host immune system, which should become activated to successfully treat neoplastic disease. Monitoring time-dependent changes in composition of tumor infiltrating population of immunocompetent cells during the treatment could help to optimize therapeutic protocols and dosage.

Water soluble polymeric conjugates based on N-(2-hydroxypropyl)-methacrylamide (HPMA) bearing anticancer drug doxorubicin bound through amide (DOX-HPMA-AM)) or hydrazone bond (DOX-HPMA-HYD) minimize unwanted side effects of conventional chemotherapy and thus enables much more aggressive treatment with very promising results. To analyze changes in tumor microenvironment during the treatment with such polymeric conjugates, we have used fine needle aspiration biopsy (FNAB). This is a commonly used clinical method to acquire samples from the body with minimal influence to tissues. It seems to be an ideal tool for gaining cell material repeatedly from one experimental animal during the treatment with simultaneous possibility to evaluate tumor growth and efficacy of used therapeutic protocol.

Materials and Methods: C57BL/6 mice bearing EL4 T-lymphoma stably expressing Enhanced Green Fluorescent Protein (EL4-EGFP+) were treated with DOX-HPMA-AM, DOX-HPMA-HYD or free doxorubicin. FNAB was applied to isolate samples from EL4-EGFP+ tumors from mice treated with different drugs. All samples were stained with specific fluorescently labeled antibodies (CD3, CD4, CD8, CD45), analyzed by flow cytometry, and verified with the samples obtained from the tumor by excision. Also the tumor progression and survival of mice after FNAB were observed regular time distances.

Results: The data obtained by FNAB or standard excision are comparable. Moreover, cell populations gained by FNAB method are more properly separated and contaminated with less debris. FNAB influences neither the tumor progression nor survival of experimental animals.

Our data show that the number of tumor-infiltrating CD4 and CD8 lymphocytes increasing rapidly on third day after treatment with DOX-HPMA-AM and DOX-HPMA-HYD, while this treatment leads to complete recovery of neoplasma in all experimental animals. Contrary to this, free doxorubicin has no influence on composition of immunocompetent cells in tumor microenvironment and also no animal was able to recover from neoplastic disease. This fact suggests that both cytotoxic and immunomobilizing effect participate on tumor regression after treatment with HPMA conjugates.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C241.
Abstract C253: Effective inhibition of Hedgehog signaling pathway with small molecule Smoothened inhibitor GDC-0449 in a bone producing prostate cancer xenograft
Authors: Karlou Maria , Tzelepi Vassiliki , Maity Sankar , Navone Nora M. , Yang Jun , Lu Jing-Fang , Hoang Anh , Logothetis Christopher J. , Efstathiou Eleni
Published: June 2012

Background: The tumor microenvironment is involved in prostate cancer progression. Several stromal-epithelial-interacting pathways associated with prostate cancer (PCa) progression in the bone, are also considered candidates of resistance to current therapeutic strategies applied. Hedgehog (Hh) signaling has been associated with PCa aggressiveness. Therefore Hh pathway could be a novel diagnostic and therapeutic target, also contributing to therapy against prostate carcinoma metastasis in bone. Experimental work on specific chemical inhibitors of Hh signaling suggests that they may represent an entirely new class of therapeutic agents. In this study we tested the efficacy of Hh pathway inhibition with Smoothened inhibitor GDC-0449 in 118b prostate cancer xenograft model.

Experimental Design: We employed the xenograft MDA PCa 118b, derived from the bone metastasis of a patient with castrate-resistant prostatic adenocarcinoma. This is characterized by unique bone producing feature when injected subcutaneously in SCID mice. Bone is the most common site for metastasis of prostate carcinoma, therefore a model with the new bone formation distinctive characteristic is really informative. Following MDA PCa 118b sc injection, animals were treated with Smoothened inhibitor GDC-0449 (oral administration, for 21 days). Hh pathway components expression was measured both in the tumor cells and stromal compartment using species specific primers for real-time PCR, western blot analysis and immunohistochemistry.

Results: Paracrine Hedgehog signaling was confirmed in the tumor xenograft microenvironment. Baseline Shh expression was present in human tumor cells and significantly higher compared to stromal compartment, while nuclear Gli1 (a hallmark of pathway activation) and Ptch1 expression in the stromal compartment were present and significantly higher compared to human tumor cells. Smoothened inhibition resulted in attenuation of stromal expression of Gli1 and Ptch1, in the treated group compared to the control thus confirming the pharmacodynamic effect of the agent. A trend for slower tumor growth at the treated group was observed, while a decline in proliferation rate was noted when Hh pathway was suppressed in the stromal compartment.

Conclusion: Hh signaling pathway demonstrates paracrine activation in the 118b prostate cancer bone producing xenograft model. GDC-0449 is effective in blocking Hh signaling in PCa. Future directions: A combination of Hedgehog signaling inhibition and cytotoxic therapies, such as androgen ablation would likely increase efficacy.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C253.
Abstract C39: Phase I clinical trial of CX-4945: A first-in-class orally administered small molecule inhibitor of protein kinase CK2
Authors: Marschke Robert F. , Andreopoulou Eleni , Von Hoff Daniel D. , Lim John K.C. , Padgett Claire S. , Northfelt Donald W.
Published: June 2012

Introduction: CX-4945 is a novel, orally administered small molecule designed to potently and selectively inhibit protein kinase CK2, a previously unexploited molecular target with well documented roles in many cancers. In vitro, CX-4945 selectively kills cancer cells by modulating key survival pathways, resulting in inhibition of proliferation, promotion of apoptosis and cell cycle arrest. Pre-clinically, CX-4945 has demonstrated single-agent potency in suppressing xenograft tumor growth with a wide therapeutic window. The objectives of this phase I study are to determine the maximum tolerated dose (MTD) and dose limiting toxicities (DLTs), to characterize the pharmacokinetics (PKs), and to study the pharmacodynamic effects of CX-4945.

Procedures: Eligible patients with advanced solid tumors, Castleman's disease or multiple myeloma with progressive disease, or for whom there are no available standard therapies, receive CX-4945 in successive dose cohorts at: 90, 160, 300, 460, 700 and 1000 mg per dose. Oral doses are administered twice daily for twenty-one consecutive days of a four week cycle. Therapy is continued in consenting patients until signs of intolerance to CX-4945 are observed, or there is evidence of advancing disease. Response by RECIST is determined after every 2 cycles. Serial blood and plasma samples are collected on the first and final dosing days of Cycle 1 (i.e., Day 1 and Day 21) for pharmacokinetic analysis and for pharmacodynamic biomarker evaluations (specifically, total and phosphorylated forms of p21 and Akt.)

Results: Thirteen patients with solid tumors (3–4 patients per cohort, from four separate dose cohorts) have received oral doses of CX-4945. These doses have been well tolerated, with no reported adverse events of grade 3 or higher. CX-4945 has demonstrated general linearity in PK parameters between the dose cohorts, with a terminal half life of approximately 25 hours at steady state.

Conclusions: To date, CX-4945 has shown no drug related toxicity and has dose proportional PKs. No DLTs have yet been observed, and the MTD remains to be defined in this Phase I study. Further enrollment to the planned dose escalation cohorts is ongoing.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C39.
Abstract C45: Preventive effect of Iressa in the hydroxybutyl(butyl) nitrosamine (OH-BBN) induced model of bladder cancer: Dose dependency, treatment timing, and alterations in biomarkers
Authors: Lubet Ronald , Bode Ann , Townsend Reid , Grubbs Clinton
Published: June 2012

The preventive efficacy of the EGFr inhibitor Iressa was examined on the induction by OH-BBN of urinary bladder cancers in female Fischer-344 rats. The OH-BBN induced model yields invasive bladder tumors with most having the histopathological appearance of transitional cell carcinomas (TCC). The resulting tumors show various protein changes; e.g., increased expression of survivin, GSTPi, and various S100 calcium binding proteins that are routinely observed in human bladder cancers. More recent array data have also implied many similarities. Recently we examined the effects of daily treatment of rats with Iressa (6 mg/kg BW/day, i.g.) beginning either 2 weeks after the last dose of OH-BBN (24 weeks of treatment) or at 12 weeks after the last dose of OH-BBN when microscopic TCCs already existed (14- weeks of treatment). Both regimens decreased the incidence of large palpable bladder cancers by 90%. This demonstrated that Iressa was highly effective even when administered late in tumor progression. In contrast, treatment with Iressa beginning 2 weeks after the last OH-BBN and followed for only 14 weeks caused a 60% decrease in the incidence of microscopic bladder cancers. Additional data examining various dose levels and the combination of suboptimal doses of Iressa with NSAIDs will be presented. In addition, we examined biomarker modulation in OH-BBN bladder cancers treated short term (5 days) with Iressa. When using a candidate marker approach, it was found that Iressa decreased levels of EGFrP as expected, but caused striking decreases in levels of phosphorylated AKT as well. A more generalized approach to modulation of biomarkers was also taken to examine altered expression of peptides in Iressa treated and control bladder tumors employing 2D gel analysis. When certain of the identified peptides were examined by pathway analysis, it was found that certain proteins associated with the VEGF pathway [ANP32A and HNPRL] were both decreased in Iressa treated tumors. Supported by NCI Contract HHSN261200433001C.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C45.
Abstract C66: Development of mechanistic assays to differentiate PI3K and mTOR inhibitors
Authors: Trinh Lan , Yen Ivana , Lesnick John , Malek Shiva
Published: June 2012

The phosphatidylinositol-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway plays a key role in the regulation of cellular growth, survival, and proliferation. Inappropriate activation of the pathway as a result of mutations and amplifications has been implicated in a variety of human cancers. Pathway dysregulation drives tumorigenesis through phosphorylation of proteins that directly regulate protein synthesis, cell cycle, and metabolism. The complexity of the mTOR and PI3K pathways, which include two functionally distinct mTOR complexes, feedback loops, and parallel regulatory pathways, clearly invokes a need for functional assays that can clarify the mechanism of action for drugs targeting mTOR and PI3K either as single agents or in combination. In this work, we used three different types of cell based assays to investigate the effects of PI3K versus mTORc1 inhibition on signaling pathways, cell cycle, and autophagy. The first set of assays differentiates an inhibitor of the mTorc1 complex versus the mTorc2 complex by utilizing two different cell lines and stimulation with either nutrient or growth factors. We show that the mTorc1 inhibitor, rapamycin, has potent activity in the nutrient stimulated mTorc1 assay, while PI3K inhibitors have very weak activity in such an assay. In the second type of assay, we used high content image analysis to produce cell cycle distribution profiles in PC3 cells, clearly showing G1 arrest for both mTOR and PI3K compounds as both single agents and in combination. However, the profiles showed no apoptotic population for either class of compounds in the cell lines tested. Therefore, as a means to investigate an alternative route of cell death to apoptosis, we also developed an autophagy assay using high content image analysis. Our results show that treatment of cells with a PI3K inhibitor or rapamycin as single agents or in combination can have different effects on autophagy and may potentially help differentiate the mechanism of action between the two classes of inhibitors. Taken together, we have generated a panel of mechanistic cellular assays which can help differentiate and characterize the effects of mTor and PI3K inhibition as both single agents and in combination.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C66.
Abstract C85: Novel molecular targets for genistein in prostate cancer cells
Authors: Merchant Kendra T. , Rathinavelu Appu , Kumi-Diaka James , Esiobu Nwadiuto , Zoeller Robert , Hartmann James , Johnson Michelle
Published: June 2012

Background: Prostate cancer is the most common form of non-skin cancer and the second leading cause of cancer deaths within the United States. The five year survival rate has increased from 69% to 99% over the last 25 years for the local and regional disease, but has remained fairly low (approximately 34%) for the advanced disease. Current research is aimed at finding complementary or alternative treatments that will specifically target components of signal transduction, cell-cycle and apoptosis pathways to induce cell death, with little or no toxic-side effects to the patient. In this study we investigated the effect of genistein on expression levels of genes involved in these pathways. Genistein (4, 5, 7-trihydroxy-isoflavone) is a major isoflavone micronutrient constituent of soy that has been shown to inhibit growth proliferation and induce apoptosis in cancer cells.

Methods: The mechanism of genistein-induced cell death and potential molecular targets for genistein in LNCaP prostate cancer cells were investigated using several techniques. The chemosensitivity of genistein towards prostate cancer cells was investigated using the ATP and MTS assays and apoptosis induction was determined using apoptosis indicator assays and caspase assays. Expression levels of several molecular targets were also determined using cDNA microarray and RT-PCR analysis.

Results: Our results revealed that genistein induces cell death in a time and dose-dependent manner and regulates expression levels of several genes involved in carcinogenesis and immunogenicity. Several cell cycle genes were down-regulated including the mitotic kinesins, cyclins and cyclin dependent kinases, indicating that genistein is able to halt cell cycle progression through the regulation of genes involved in this process. Several members of the Bcl-2 family which are involved in apoptosis were also affected and a number of genes involved in immunogenicity were upregulated including DefB1 and HLA membrane receptors.

Conclusion: The results of this study provide evidence of genistein's ability to inhibit cancer cell proliferation and induce apoptosis which indicates its potential as an adjuvant in chemotherapy and immunotherapy.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C85.
Abstract C94: Deubiquitylases as novel anticancer targets: Discovery and development of novel USP7 inhibitors
Authors: Kumar KG Suresh , Cao Ping , Goldenberg Seth J. , Leach Craig A. , McDermott Jeffrey L. , Kodrasov Matthew P. , Chauhan Dharminder , Tian Ze , Anderson Kenneth C. , Mattern Michael R. , Nicholson Benjamin
Published: June 2012

The ubiquitin-proteasome pathway is a complex system consisting of enzymes that conjugate or deconjugate ubiquitin to/from target proteins upstream of the 26S proteasome. In particular, ubiquitin deconjugation is performed by families of proteases known as deubiquitylating enzymes (DUBs). The therapeutic validation of the ubiquitin-proteasome system was provided by the marketing approval of bortezomib (PS-341) for the treatment of multiple myeloma (MM). In our search for alternative approaches for the treatment of MM, we investigated the therapeutic potential of inhibiting another component of the ubiquitin-proteasome pathway, the DUB USP7. Our rationale for targeting USP7 is as follows: 1) Blockade of USP7 using siRNA or homologous recombination results in elevated levels of the tumor suppressor p53 and cell cycle arrest in cancer cells. In contrast to the situation in the majority of human tumors, mutations or deletions of p53 in MM are relatively rare and thus the activation of p53 is likely to offer a therapeutic benefit in MM. 2) Knockdown of USP7 destabilizes the adaptor protein Claspin, inhibiting activation of Chk1, an effector kinase in the DNA damage response, abrogating the G2M cell cycle checkpoint and providing a mechanism for inducing tumor cell death in a p53 independent manner. Thus inhibition of USP7 would be predicted to activate pro-apoptotic and inactivate pro-survival pathways in multiple myeloma, resulting in tumor suppression.

Recently, using its proprietary screening technology, Progenra discovered and characterized a small molecule inhibitor of USP7, P5091. Specifically, P5091 selectively inhibits USP7 relative to other proteases and exhibits cytotoxic activity against both p53 wild type and p53 mutant tumor cell lines. Furthermore, P5091 destabilizes the pharmacodynamic markers HDM2 and Claspin in tumor cells and induces cell death synergistically in combination with genotoxic agents. Medicinal chemistry has identified a series of potent and selective novel chemical entities. Initial preclinical studies at the Dana-Farber Cancer Institute demonstrate that P5091 induces apoptosis and is cytotoxic against human MM cells but not normal plasma cells.

In conclusion, these data demonstrate that 1) USP7 is a viable target for the treatment of MM and other cancers; and 2) P5091 is an efficacious inhibitor of USP7. Preclinical development of the P5091 series continues, augmented by a comprehensive medicinal chemistry program designed to identify a clinical candidate from this series in 2010.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C94.
Abstract C95: Role of 5-alpha reductase inhibitor in regulating ETS transcription factors in prostate cell lines
Authors: Ateeq Bushra , Vellaichamy Adaikkalam , Chiannaiyan Arul M. , Varambally Sooryanarayana
Published: June 2012

Androgens play a crucial role in normal and cancerous growth and hence the androgenic pathway has become an important target of therapeutic intervention. We have discovered the gene fusions of the 5′-untranslated region (UTR) of androgen regulated gene TMPRSS2 (21q22.3) with the ETS transcription factor family members, either ERG (21q22.2), ETV1 (7p21.2), or ETV4 (17q21), in the majority of prostate cancers. This provides a mechanism for over-expression of ETS genes leading to tumor progression. Dutasteride (Avodart®) is a 5-alpha-reductase inhibitor that blocks the synthesis of the intracellular androgen dihydrotestosterone (DHT).

Here we tested the effect of dutasteride on TMPRSS2-ERG fusion positive VCaP cells. Our data showed 5-alpha-reductase SRD5A1 and SRD5A3 isoenzymes are highly expressed in metastases as compared to benign and prostate cancer cases. On the contrary, SRD5A2 is highly expressed in benign and prostate cancer cases. We found dutasteride treated VCaP cells at 10 and 50uM concentration showed a significant decrease in cell proliferation along with distinct morphological changes. Cells treated with synthetic androgen R1881, testosterone or DHT showed a drastic increase in invading cells, while dutasteride at 50uM concentration showed a significant decrease in cell invasion alone or in combination with testosterone or DHT.

VCaP cells grown under androgen deprivation or treated with 10uM dutasteride showed reduction in ERG and PSA expression. Conversely, cells treated with testosterone or DHT resulted in a marked induction of PSA and ERG as anticipated, while in cells pre-treated with dutasteride, there was a further decrease in the ERG and PSA expression. Addition of testosterone to cells treated with dutasteride slightly increased the ERG expression. Our data clearly suggest that inhibition of 5-alpha reductase by dutasteride significantly reduces the fusion gene expression.

We further tested our hypothesis in an in vivo VCaP-luciferase xenograft model. We found bicalutamide alone showed a decrease in tumor volume in VCaP-Luc xenografts, whereas the combination of dutasteride and bicalutamide reduced tumor burden significantly. Our findings provide insights into the function of dutasteride within the tumor microenvironment, potentially allowing for development of therapeutics that can be used in combination with this drug to further enhance its effectiveness in ETS positive prostate cancer.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C95.
Abstract C99: Panitumumab conjugated gold nanoparticles induce hyperthermic cytotoxicity in pancreatic carcinoma cell lines after noninvasive radiofrequency field exposure
Authors: Glazer Evan S. , Curley Steven A.
Published: June 2012

Introduction: Many cancer cells that highly express epidermal growth factor receptor-1 (EGFR-1) have been demonstrated to endocytose antibody specific targeted nanoparticles such as cetuximab conjugated gold nanoparticles. These gold nanoparticles are subsequently delivered throughout the cell. When gold nanoparticles are exposed to near infrared laser (NIR) radiation or non-invasive radiofrequency fields, they heat in a concentration dependent manner. Radiofrequency (RF) fields are advantageous because they easily penetrate through body tissues without injury. We investigated the effects of a clinically available EGFR-1 antibody, panitumumab, conjugated to gold nanoparticles in noninvasive RF fields.

Methods: Solid gold nanoparticles (10 nm) were covalently linked to panitumumab, a monoclonal antibody against EGFR-1. Two pancreatic carcinoma cell lines, Panc-1 and Capan-1, were treated with 200 nM panitumumab linked gold nanoparticles in media for four hours. After replacing the media with fresh media not containing the antibody linked gold nanoparticles, the cells were exposed to an RF field at a generator power of 600W for a total of 3 minutes. The media temperature was kept below 42°C in order to prevent non-specific apoptosis. Viability and apoptosis were evaluated with flow cytometry three days later. Statistical analysis was performed with the Student's t-test and uncertainties represent standard errors of the mean.

Results: The viability of control cells not treated with the conjugate remained greater than 90% for both cell lines. The antibody-nanoparticle conjugate alone decreased the viability of Panc-1 cells to 81.0% ± 1.1%. However, after RF field exposure, the viability decreased to 57.2% ± 7.9% (p < 0.03). After antibody-conjugate treatment, Capan-1 cells viability was 89.5% ± 3.9%. After RF field exposure, however, the viability decreased to 67.8% ± 3.8% (p <0.03).

Conclusions: The susceptibility of pancreatic carcinoma cell lines to gold nanoparticle mediated noninvasive RF field hyperthermic therapy may be related to the combined effects of monoclonal antibody treatment and intracellular hyperthermia. Based on this simple model, it appears that there is a relationship between EGFR-1 expression, cytotoxicity associated with monoclonal antibody therapy, and hyperthermic cytotoxicity after RF field exposure. Combined rational therapy utilizing non-invasive RF fields may provide for opportunities where traditional radiotherapy has failed and NIR therapy is not readily possible due to tumor location deep within a body cavity.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C99.