bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract B103: HSP90 inhibition targets the dysregulated signal and transcription factor network and malignant phenotype of head and neck squamous cell carcinoma.
Authors: Gouveia Christopher J. , Wise Stephanie , Friedman Jay , Hu Michael , Freudlsperger Christian , Kannabiran Vishnu , Arun Pattatheyil , Mitchell James , Kan Julie , Brega Nicolleta , Chen Zhong , Van Waes Carter
Published: June 2012

Purpose: Activation of multiple oncogenic signaling pathways and transcription factors, concurrent with defective tumor suppressor TP53, have been implicated in pathogenesis and therapeutic resistance of epithelial cancers. Many proteins upon which these cancers depend are stabilized by the chaperone, heat shock protein 90 (HSP90). Here we characterized the effects of HSP90 inhibitor, PF-04928473, against the dysregulated network and malignant phenotype in head and neck squamous cell carcinoma (HNSCC).

Experimental Design: HSP90 expression was analyzed in HNSCC tissue array and cell lines. Expression and function of oncogenic molecules and cytokines were analyzed by Western blot, reporter gene, and multiplex Luminex assays. Drug effects on proliferation, cell cycle, and radiosensitivity were evaluated by MTT, flow cytometry, and clonogenic assays. Anti-tumor effects and modulation of immunohistochemical markers were evaluated in a TP53-deficient xenograft model resistant to targeted therapies.

Results: HNSCC tissues and lines overexpressed HSP90. PF-04928473 inhibited dysregulated EGFR, c-MET, AKT, ERK1/2, IKK signal molecules, AP-1, STAT3, and NF-B reporter genes, and expression of target oncogenes and angiogenic cytokines. PF-04928473 induced re-expression of TP53 and targets p21 and PUMA in TP53-deficient HNSCC. Corresponding to effects on these molecules implicated in HNSCC pathogenesis, PF-04928473 inhibited proliferation, induced G2/M block, apoptosis, and enhanced radiosensitivity in vitro. PF-04928473 similarly modulated key targets, inducing apoptosis, and inhibiting proliferation, angiogenesis and xenograft tumorigenesis in vivo.

Conclusion: HSP90 antagonist PF-04928473 modulated the dysregulated signal and transcriptional network, inhibited the malignant phenotype, and radiosensitized HNSCC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B103.
Abstract B121: Influence of affinity tags on the biochemical properties of recombinant human tyrosine kinase ALK.
Authors: Mueller Daniel , Totzke Frank , Beisenherz-Huss Christian , Heidemann-Dinger Caroline , Weber Thomas , Ketterer Constance , Kubbutat Michael H.G.
Published: June 2012

Anaplastic lymphoma kinase (ALK) is a human receptor tyrosine kinase which has important functions in the development and maintenance of the peripheral and central nervous system. It has also been shown to be one causative agent in the development of several human malignancies including neuroblastoma, anaplastic lymphoma and non small cell lung cancer (NSCLC). One mechanism of pathological activation of ALK occurs by chromosomal translocation of the ALK gene to EML4 giving rise to an abnormal fusion protein. In order to develop small molecule inhibitors targeting ALK, in-vitro kinase assays have been developed which use recombinant ALK fragments fused to affinity tags to facilitate purification. Here we present data which demonstrate that biochemical parameters of recombinant ALK fusion proteins, like substrate specificity, ATP Km, Vmax and effects of chemical compounds, are significantly influenced by the type of affinity tag used. That influence partially persists even after cleavage of the affinity tag during the purification process. Since affinity-tagged recombinant proteins are widely used in drug discovery, a careful evaluation of potential tag-related effects should be considered.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B121.
Abstract B144: Elesclomol, an inducer of oxidative stress, targets oxidative phosphorylation (oxphos) in melanoma cells.
Authors: Moschos Stergios J. , de Moura Michelle Barbi , Fayewicz Shelley , Bateman Nicholas , Sun Mai , Duensing Stefan , Yin Yan , Sander Cindy , Becker Dorothea , Kirkwood John , Conrads Thomas , Van Houten Bennet
Published: June 2012

Mitochondria are important gatekeepers of cell survival in melanoma cells and their role as oxygen sensors, ATP producers, or inducers of reactive oxygen species are established. However, the role(s) of metabolic processes other than glycolysis are not well characterized in melanoma development and progression. Here we show that melanoma cells exhibit higher levels of oxidative phosphorylation (Oxphos) compared to human epidermal melanocytes. Furthermore, we present several independent lines of evidence (bioenergetics analysis using Seahorse XF24, stable isotope labeling of melanoma cells in culture, immunoblot analysis of isolated mitochondria, electron microscopy, and rho zero cells) that elesclomol, an inducer of oxidative stress, suppresses Oxphos in melanoma cells without a significant effect on glycolysis in vitro. Melanoma cells with intrinsically high levels of glycolysis are less sensitive to elesclomol. In addition, following chronic continuous drug exposure, resistant cells manifest a high glycolytic phenotype. Finally hypoxia inducible factor 1 alpha, a master metabolic switch of cell metabolism towards glycolysis, is upregulated following elesclomol treatment in a number of melanoma cell lines examined. Our results suggest that Oxphos is an important therapeutic target and that elesclomol specifically impinges on this process.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B144.
Abstract B149: Yes-associated protein (YAP) is associated with aggressive characteristics of BRAFV600E thyroid cancer.
Authors: Lee Seong Eun , Lee Jung Uee , Lee Min Hee , Ryu Min Jeong , Kim Soung Joong , Kim Yong Kyung , Kim Koon Soon , Choi Min Jeong , Jo Young Suk , Shong Minho
Published: June 2012

Up regulation of Yes-associated protein (YAP), a transcriptional co-activator regulated by Hippo (MST-LAST-YAP) pathway, has been reported to mediate oncogenic transformation in animal models of hepatocellular carcinomas. However, the expression status and molecular biological role of YAP in thyroid cancer remained to be investigated. We evaluated YAP expression and identified increased nuclear YAP expression in PTC and ATC. Interestingly, we could observe nuclear YAP staining more frequently in BRAFV600E(+) PTC. We also performed immunofluorescence staining to verify intracellular localization of YAP in TPC1 (harboring RET/PTC1), 8505C (harboring BRAFV600E) and BRAFV600E stably transfected HEK293T cells. Consistently, we could detect nuclear YAP in 8505C and BRAFV600E expressed cells, but not in TPC1. Although cell viability assays did not show any difference between YAP knockout cells and control cells generated by using shYAP in 8505C and shCTL (control) 8505C, YAP knockout cells showed remarkably decreased migration ability in the scratch assays. Using orthotopic mice models, shYAP 8505C injected mice showed decreased local invasion ability and less frequent lung metastasis. In conclusions, Nuclear YAP could be more frequently detected in BRAFV600E(+) thyroid cancer compared to BRAFV600E(−) thyroid cancer. Furthermore, nuclear YAP in BRAFV600E(+) thyroid cancer was associated with local invasiveness and distant metastasis, presenting that YAP could affect the aggressive phenotype of BRAFV600E(+) thyroid cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B149.
Abstract B15: The use of micro-CT imaging and histology in a clinically relevant Foxn1nu mouse intrafemoral model of metastatic prostate cancer.
Authors: Batey Michael A. , Brown Matthew , Booth Catherine
Published: June 2012

Subcutaneous tumor xenograft models using transplantation of human tumour cell lines into immunodeficient mice are routinely used for the preclinical testing of anticancer therapies. These models do not however fully represent the clinical situation, and as such there can be difficulties in translating the results of promising preclinical data into a successful patient treatment. There is therefore a need to develop more clinically relevant preclinical models, particularly in metastatic disease. The development of preclinical imaging strategies, such as micro-CT gives us the ability to follow disease progression at orthotopic sites, and the efficacy of treatment longitudinally in a similar way to the clinical situation.

At autopsy, approximately 84% of patients with prostate cancer will be found to have skeletal metastases, and in those patients who first present with bony disease the 5 year survival rate is less than 25%, making this an important focus for the development of novel therapies. Intrafemoral (i.f.) transplantation is a technically feasible and convenient method for the study of cancer types associated with bone or bone marrow, as this technique implants cells directly into the biological niche of interest, correlating with the environment in which disease develops in patients.

Previously, severely compromised immunodeficient mice have been used to study metastatic prostate cancer development. For the studies described here, using the immunocompromised Foxn1nu mouse strain, the human prostate cancer cell lines PC3 (prostate membrane specific antigen negative, androgen resistant) or LNCaP (prostate membrane specific antigen positive, androgen sensitive) were injected i.f. into the bone marrow cavity of the femur via the knee joint. Animals were followed over a period of several weeks, and micro-CT imaging used to assess the growth and development of tumour in bone. Areas of interest, identified by imaging, were taken for histological assessment of patient relevant markers, and data compared to clinical imaging and histopathology of human disease.

We have found that this model offers a clinically relevant preclinical representation of bony metastasis of prostate cancer. The described use of micro-CT imaging and detailed histological analysis allows for a clearer translation between preclinical testing and clinical observation, and as such represents a valuable new tool for the assessment of novel therapeutics.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B15.
Abstract B161: PIK3CA mutations H1047R are associated with response to PI3K/AKT/mTOR signaling pathway inhibitors in early-phase clinical trials.
Authors: Janku Filip , Naing Aung , Stepanek Vanda T. , Falchook Gerald S. , Hong David S. , Tsimberidou Apostolia M. , Fu Siqing , Lee J. Jack , Luthra Rajyalakshmi , Wheler Jennifer J. , Kurzrock Razelle
Published: June 2012

Background: Activating mutations in the PIK3CA gene have been identified in many malignancies. Preclinical and early clinical data suggest that these mutations predict response to PI3K/AKT/mTOR inhibitors in subsets of patients. Sensitivity and resistance mutation have not been reported to date.

Methods: Patients with diverse cancers referred to the Phase I Clinical Trials Program for targeted therapy starting from October 2008 were analyzed for PIK3CA, and if, possible for KRAS mutations using PCR-based DNA sequencing. Patients with any tumor type and any PIK3CA mutation were treated whenever possible with agents targeting the PI3K/AKT/mTOR signaling pathway. Treatment outcomes with respect to mutation types were analyzed.

Results: Overall, 1012 patients were tested and 105 (10%) had PIK3CA mutations. Of the 105 patients with PIK3CA mutations, 66 (median number of prior therapies, 3) were treated on a protocol that included a PI3K/AKT/mTOR pathway inhibitor. Of these 66 patients, 11 (17%) achieved a partial response (PR) and 4 (6%) stable disease ≥ 6 months (SD≥6). The total of SD≥6/PR was 15 (23%). Patients with PIK3CA H1047R mutation (n=16) compared to patients with other PIK3CA mutations (n=50) had higher PR rate (6/16, 37.5% vs. 5/50, 10%; p=0.018), higher rate of SD≥6/PR (7/16, 44% vs. 8/50, 16%; p=0.037), and trend toward longer median progression-free survival (PFS) (5.7 months, 95%CI 0.4–11.0 vs. 2.0 months, 95%CI 1.8–2.2; p=0.064). Patients with a PIK3CA E542K mutation (n=11) compared to others (n=55) trended toward a worse median PFS (1.8 months, 95%CI 1.4–2.2 vs. 2.6 months, 95%CI 1.4–3.8; p=0.058). Of the 66 treated patients with PIK3CA mutations, 55 (83%) had enough tissue for KRAS testing. None of the 16 patients with PIK3CA and simultaneous KRAS mutations in codon 12 or 13 had a PR (0/16, 0% vs. 9/39, 23%; p=0.046) or SD≥6/PR (0/16, 0% vs. 13/39, 33.3%; p=0.011) as compared to patients with PIK3CA, but not KRAS codon 12 or 13 mutations, whereas 2 of 2 (100%) patients with PIK3CA and Q61H KRAS mutation in codon 61 had SD≥6 (n=1) and PR (n=1).

Conclusion: Despite the small number of patients, our data suggest that PIK3CA mutations H1047R are associated with response to PI3K/AKT/mTOR inhibitors, whereas codon 12 and 13, but not codon 61 KRAS mutations are associated with resistance.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B161.
Abstract B186: Knockdown of a chromatid segregation mediator gene impairs tumor growth in glioblastoma multiforme.
Authors: Mughal Awais , Stangeland Biljana , Ramsnes Ingunn , Grieg Zanina , Joel Mrinal , Fjerdingstad Hege , Sandberg Cecilie , Vik-Mo Einar Osland , Murrell Wayne , Langmoen Iver Arne
Published: June 2012

Glioblastoma Multiforme (GBM) is the most common primary brain malignancy and despite multimodal therapy including surgery, irradiation and chemotherapy median survival remains 12–15 months. Increasing body of evidence from our and other groups is confirming the existence of a stem-like cell population within these tumors believed to confer tumor growth, invasiveness, and therapy resistance. Adult human neural stem cells (aHNSCs) are suggested to be the cells of origin for the glioblastoma stem-like cells (GSCs).

To identify new molecular targets we performed a micro-array analysis on the transcriptome of 33000 genes identifying differentially expressed genes in 5 aHNSC and 9 GSC cultures. Amongst the genes highly up-regulated in GBM, several were involved in cell division and the regulation of cell cycle. For further analysis, we chose the Chromatid Segreration Mediator (CSM), a centromere specific gene important for appropriate chromatid segregation during mitosis. This candidate gene was strongly expressed in GSCs but was almost undetectable in aHNSCs. Abundance of the CSM protein may lead to aneupleudy, which is a common feature of GBMs.

The high levels of CSM mRNA were confirmed by analyzing 16 GSC cultures with micro-array and qPCR. CSM was also over-expressed on protein level, and was found to be highly immunopositive in glioblastoma biopsies compared to the normal human brain. In silico analysis, using Rembrandt, identifies CSM-gene over-expression as a negative prognostic indicator in glioma patients.

In order to study the functional role of this gene in GBM, we used lentiviral-based knock-down (KD) technology to obtain specific down regulation of the gene. We transduced different shRNA constructs in 2 GBM cultures and achieved KD-efficieny of 55–65%.

Growth of tumors was measured by proliferation assay and the relative number of sphere-forming cells (widely known as GSCs) was quantified with a sphere-formation assay.

In our KD-cultures we observed a moderate reduction of proliferation (25%). Interestingly, the sphere forming capacity was dramatically reduced by 60–70%, indicating a significant decrease in number of GSCs. Cell-cycle analysis revealed that it was 30–40% less cells in G2/M-phase and 15–20% more cells in G0/G1-phase in the KD compared to non-silencing control.

We find our results very promising and suggest that Chromatid Segregation Mediator gene might have potential as a novel therapeutic target for GBM management.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B186.
Abstract B187: ErbB4 ectodomain as a biomarker and a potential therapeutic target for breast cancer.
Authors: Hollmén Maija , Liu Ping , Wildiers Hans , Reinvall Irene , Vandorpe Thijs , Smeets Ann , Deraedt Karen , Vahlberg Tero , Joensuu Heikki , Leahy Daniel J. , Schöffski Patrick , Elenius Klaus
Published: June 2012

ErbB4 function in breast cancer is controversial and influenced by alternative splicing of the ERBB4 gene. Here we evaluated the cleavable ErbB4 JM-a isoform as a potential drug target and biomarker. Matched serum and tissue samples from a series of 243 breast cancer patients were analyzed for ErbB4 cleavage products by ELISA and immunohistochemistry. Elevated serum ectodomain level (≥40 ng/mL) was present in 21% of the patients and significantly associated with the premenopausal status at diagnosis (P = 0.04). Consistent with the in vivo data, estradiol enhanced ErbB4 shedding and TACE activity in vitro. Selective targeting of ErbB4 JM-a by mAb 1479 inhibited ErbB4 cleavage and reduced tumor formation in a mouse xenograft model. The 3.4 Å X-ray crystal structure of the sErbB4:1479Fab complex localized the binding site of mAb 1479 on ErbB4 to a region on subdomain IV encompassing JM-a-specific residues. These data demonstrate that ErbB4 cleavage can be specifically targeted in vivo, and introduce serum ErbB4 ectodomain concentration as a biomarker for monitoring the activity of compounds that target ErbB4 cleavage.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B187.
Abstract B199: Identification of novel tubulin polymerization inhibitors by proteomic profiling.
Authors: Kawatani Makoto , Tomita Kouji , Muroi Makoto , Kazami Sayaka , Aono Harumi , Shimizu Takeshi , Osada Hiroyuki
Published: June 2012

Background: Every year huge numbers of compounds are screened for cancer cells proliferation inhibition. However, the mechanism of action and/or the molecular target as decisive factors in drug development often remain unknown. We recently developed a proteomic profiling technique to predict the mode of action of new drug leads. This method is based on recording the comprehensive patterns of variation in proteins in HeLa cells that are treated with compounds. The primary in vitro screening of the candidate anticancer compounds from the chemical library of RIKEN Natural Products Depository (NPDepo) revealed potentially active compounds with diverse chemical structures (NPD6689, NPD8617, and NPD8969). Here, we report on a rapid identification of a molecular target for these compounds by using the proteomic profiling system.

Materials and Methods: HeLa cells were treated with NPD6689, NPD8617, or NPD8969 for 18 h, and the cell lysates were subjected to 2-dimensional fluorescence differential gel electrophoresis (2D-DIGE). The proteome data were linked to our database, which contained proteome data that were generated by 47 compounds with well-established modes of action. Then, hierarchical cluster analysis was performed.

Results: NPD6689, NPD8617, and NPD8969 inhibited the growth of HeLa cells with the IC50 value of 615, 184, and 240 nM, respectively. The proteomic profiling of the effects induced in HeLa cells by 2D-DIGE revealed the NPD6689, NPD8617, and NPD8969 to belong to the same cluster as tubulin inhibitors, like vinblastine, paclitaxel, and colchicine. The predicted mode of action was confirmed by direct inhibition of tubulin polymerization, disruption of microtubule network in interphase cells, abnormal spindle morphology in mitotic cells, and cell cycle arrest at G2/M phase. The effects were time- and concentration-dependent.

Conclusion: These results indicate that NPD6689, NPD8617, and NPD8969 target tubulin and act as its polymerization inhibitors. Furthermore, our study shows that proteomics-based profiling will be very useful in rapid identification of mechanisms of action of anticancer compounds in future drug development and chemical genetic studies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B199.
Abstract B2: Regorafenib, an oral multi-kinase inhibitor of angiogenic, stromal and oncogenic receptor tyrosine kinases, potently inhibits tumor growth and angiogenesis in patient-derived xenograft models of gastric cancer.
Authors: Hung Huynh T. , Ong Richard , Soo Khee Chee , Politz Oliver , Zopf Dieter
Published: June 2012

Background: Gastric cancer (GC) is a prevalent and deadly disease. Because the VEGFR and PDGFR signaling cascade plays a critical role in the development and progression of gastric cancer and VEGF expression is correlated with angiogenesis, tumor progression and poor prognosis in patients with GC, blocking these signaling pathways could thus have therapeutic efficacy. This study aims to investigate the potential of a combined therapeutic approach to target these pathways in preclinical models of GC with regorafenib an oral multi-kinase inhibitor targeting VEGFR1–3, PDGFR, FGFR, TIE-2, RET and KIT.

Methods: Eight patient-derived GC xenograft models with different histotypes were treated orally with regorafenib or vehicle and tumor growth was monitored using caliper measurements. Tissue sections of vehicle- or regorafenib-treated xenografts were analyzed by immunohistochemistry for apoptosis, microvessel area, and cell proliferation using antibodies against cleaved PARP, CD31 and phospho-Histone3Ser10, respectively. Tumor lysates were analyzed by Western Blotting with respective antibodies for effects on marker proteins for proliferation, apoptosis and angiogenesis.

Results: Regorafenib demonstrated significant tumor growth inhibition of 81%, 85% and 88% in the GC09-0109 model after oral administration of 5, 10 and 15 mg/kg/d, respectively. In addition potent tumor growth inhibition ranging from 72–96% was observed in seven other GC models at a dose of 10 mg/kg/d. Regorafenib significantly inhibited angiogenesis in all models by 4–12 fold compared to the vehicle-treated group, independent of their degree of vascularization, as indicated by reduced CD31 staining. Furthermore inhibition of cell proliferation by 2–4 fold was observed by reduced staining for p-histone 3Ser10. Increased staining for caspase-cleaved p85 PARP by 2 to 20 fold was detected, indicating that regorafenib induced apoptosis. Finally, regorafenib also induced tumor necrosis as detected by histological staining. Inhibition of angiogenesis, reduced cell proliferation and induction of apoptosis and necrosis was associated with decreased levels of p-ERK1/2, p-cdk2Thr14/Tyr15, p-VEGFR2Tyr951, p-p70S6KThr421/Ser424, p-S6RSer235/236 and p-eIF4ESer209 and elevation of cleaved caspase-3 and cleaved PARP.

Conclusion: We show that regorafenib is an efficacious anti-tumor targeted agent in patient-derived gastric cancer preclinical models. Our findings support further investigation of regorafenib in the treatment of GC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B2.
Abstract B213: Isolation of muscle fatigue substance(s) with antitumor activities.
Authors: Munoz Ruben M. , Han Haiyong , Von Hoff Daniel D. , Hoffman Stanley
Published: June 2012

It has been scientifically established that regular physical exercise can reduce cancer risk by as much as 40% (Newton et al. 2008 Curr Treat. Options Oncol. 9:135). In early 1960's, Hoffman and colleagues reported that electric stimulation of rat femoris muscles caused the release of certain substance(s) that inhibited growth of transplanted rat tumors. The researchers called the substance “F-Substance” (fatigue substance) (Hoffman et al. 1961 Cancer Res. 22:597). However, the nature of the substance and the mechanism of its action are not known. In this study we sought to isolate and characterize the F-Substance. Femoris muscles freshly removed from 8–9 week old Wistar rats were electrically stimulated to induce contraction fatigue in a saline solution. Substances released to the saline solution were dialysed and lyophilized. Cell growth assays showed the substances isolated from the stimulated muscles had more significant inhibitory effect on the breast cancer cell line MCF-7 that those isolated from unstimulated muscles. To identify the molecule(s) responsible for the antitumor activity, we used a rat cytokine antibody array to profile the cytokines in the substances. Among the 29 different cytokines contained on the array, 3 showed greater than 3-fold difference between the substances isolated from the stimulated and unstimulated muscles. LIX (also known as CXCL5) is 6-fold higher in the substances isolated from stimulated muscles than those from the unstimulated muscles. TIMP-1 is 4.6 fold higher and sICAM-1 is 3.6 fold higher in the substances from the stimulated muscles. We are currently confirming the results by Western blotting and also evaluating the antitumor activity of these cytokines using cell based assays. Our results indicated that cytokines released from contracting muscles might be responsible for the antitumor effect of the “F-Substance”.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B213.
Abstract B22: Next-generation markers and targets in breast cancer: an integrative omics approach.
Authors: Barh Debmalya , Tiwari Sandeep , Bhat Dattatraya
Published: June 2012

Introduction: Although the complete human genome sequence and ultra high throughput screening (HTP) techniques have provided us a large number of Omics based potential molecular markers and drug targets, till date, most of these markers and targets fail to pass the initial phases of clinical trials (CTs) for any given cancer creating a big obstacle in developing personalized anticancer therapeutics. Therefore, a new strategy is required to analyze these Omics data and subsequent development of superior markers and targets. This study was aimed to explore molecular insights and to identify next-generation targets and targeted therapy in breast cancer (BC) based on an integrative omics approach.

Methods and Results: Using various Omics data for BC and an integrative bioinformatics strategy, we found that [1] there are more than one cross talking signalling pathways so the targets present in most BC cases. Therefore Herceptin like single targeting (HER2) drug alone cannot be 100% effective. [2] Four to five key transcription factors (TFs) directly regulate key tumorigenic pathways. [3] These TFs also regulate their targets through a number of key miRNAs. [4] Using feedback loop regulation, these miRNAs also regulate their regulatory TFs. [5] Such TFs and miRNAs can also regulate cancer stem cell micro-environment via onco-fetal oncogenes and pluripotent stem cell factors using similar feedback loop mechanism. [6] Identified TFs and miRNAs are differentially expressed in BC and may be used as early diagnostic and prognostic markers. [7] Upregulated TFs and miRNAs may be used as superior drug targets and [8] downregulated miRNAs may be considered as candidate therapeutics.

Discussion: Our integrative analysis demonstrates that heterogeneous signalling cross talks are prevalent in BC aetiology. Therefore, patient specific drug targets should be identified and multi-targeting approach must be recommended. Similarly, identified new markers and targets are required to be validated for their efficacy in CTs and in developing next generation targeted therapeutics.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B22.
Abstract B224: Structure-based drug design of signal transducers and activator of transcription 5 inhibitors.
Authors: Cumaraswamy Abbarna A. , Gunning Patrick T.
Published: June 2012

Signal Transducer and Activator of Transcription 5 (Stat5) protein has gained notoriety for its aberrant role in many human cancers including breast, liver, prostate, skin, head and neck. Despite the significant evidence showing Stat5's role in cancers, there has been little progress in developing small molecule inhibitors of Stat5 function. In contrast, there has been significant advancements in identifying inhibitors for the Stat3 protein. As a result, several potent and direct small molecule and oligonucleotide-based inhibitors have entered pre-clinical trials as a Stat3 therapeutic. Unfortunately, there has not been progress towards the development of potent Stat5 inhibitors.

Stat5 is activated by ligand-receptor binding, which recruits Stat5 to intracellular receptor sites via their SH2 domain. Stat5 is then phosphorylated at conserved tyrosine (Y) residues Y694 and 699, which facilitates the formation of active Stat5 dimers via reciprocal pY-SH2 domain interactions. The dimers then translocate to the nucleus and induce target gene transcription. In cancer cells, Stat5 is constitutively phosphorylated leading to the aberrant expression of anti-apoptotic Stat5 target genes.

In an effort to identify direct inhibitors of Stat5, we conducted an in vitro screen of a focused library of SH2 domain binding salicylic acid-containing inhibitors (∼150) against Stat5. Several potent (Ki < 5 μM) and Stat5 selective (>3-fold specificity for Stat5 cf. Stat1 and Stat3) inhibitors were identified which were then evaluated in K562 and MV-4–11 human leukemia cells which are known to harbour constitutively activated Stat5. BP-1–108 was identified as the most potent lead within these cell lines. The lead was further evaluated to show potent induction of apoptosis (IC50's ∼ 20 μM) which correlated with potent and selective suppression of Stat5 phosphorylation. In addition, it inhibited Stat5 target genes; Cyclin D1 & c-myc. BP-1–108 also showed no cytotoxicity against healthy bone marrow cells at concentrations up to 160 μM. These inhibitors represent the most potent and direct inhibitors of Stat5 function reported to date. An in silico docking analysis with BP-1–108 within the Stat5b crystal structure (pdb:1Y1U) revealed a tertrapodal projection of functionality facilitating access to a number of sub pockets within the Stat5 SH2 domain. Currently we are conducting a structure-activity relationship against Stat5's SH2 domain with one projection into the hydrophobic pocket using diverse heterocycles to delineate inhibitor binding to improve the potency of our inhibitors.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B224.
Abstract B235: Characterization of LY2228820 dimesylate, a potent and selective inhibitor of p38 MAPK with antitumor activity.
Authors: Campbell Robert M. , Anderson Bryan D. , Chan Edward M. , De Dios Alfonso , Gilmour Raymond , Hatch Steven D. , Jambrina Enrique , Lee Paul , Mader Mary , Na Songqing , Neubauer Blake L. , Pratt Susan , Shih Chuan , Stancato Louis F. , Starling James J. , Velasco Juan A. , Watkins Scott A. , Wolos Jeffrey A. , Ye Xiang
Published: June 2012

p38α mitogen-activated protein kinase (MAPK) is activated by cancer cells in response to environmental factors, such as oncogenic stress, radiation, and chemotherapy. p38α MAPK phosphorylates a number of substrates, including MAPKAP-K2 (MK2), and regulates the production/message stability of cytokines produced in the tumor microenvironment, such as TNFα, IL1 β, IL-6 and IL-8. p38α MAPK is activated and highly expressed in human cancers and may play a role in tumor growth, invasion and metastasis. LY2228820 dimesylate is a tri-substituted imidazole derivative that is a potent and ATP-competitive inhibitor of the α and β isoforms of p38 MAPK in vitro (IC50 = 5.3 nM and 3.2 nM, respectively). This compound displays > 1000-fold selectivity for p38α MAPK versus 179 other kinases tested (including p38δ and γ isoforms). In cell-based assays, LY2228820 dimesylate also potently and selectively inhibits phosphorylation of MK2 (Thr334) in TNFα-stimulated HeLa cells (IC50 = 8.1 nM) and anisomycin-induced mouse RAW264.7 macrophages (IC50 = 35.3 nM) with no changes in phosphorylation of p38α MAPK, JNK, ERK1/2, c-jun, ATF2 or cMyc at concentrations up to 10μM. LY2228820 dimesylate also reduces TNFα secretion by LPS/IFNγ-stimulated macrophages (IC50 = 6.3 nM). In mice transplanted with B16-F10 melanomas, phospho-MK2 was effectively inhibited by LY2228820 dimesylate in tumors in a dose-dependent manner (TMED70 = 19.4 mg/kg). Significant target inhibition (>40% inhibition of phospho-MK2) was maintained for approx. 4–8hrs following a single 10mg/kg oral dose. In a broad range of xenograft models (A-549 NSCLC, SK-OV-3 Ovarian, U-87MG Glioma, MDA-MB-468 Breast), LY2228820 dimesylate demonstrates significant tumor growth delay. In summary, LY2228820 dimesylate is a novel, potent and selective inhibitor of p38 MAPK with anti-tumor activity. Further studies are ongoing to determine its molecular mechanism(s) of action, potential for combination with standard-of-care agents, and potential clinical activity.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B235.
Abstract B246: NVP-BGJ398: A potent and selective inhibitor of the fibroblast growth factor receptor family.
Authors: Guagnano Vito , Porta Diana Graus , Bordas Vincent , Douget Mickael Le , Stamm Christelle , Furet Pascal , Spanka Carsten , Wartmann Markus , Schmid Herbert , Brueggen Josef , Drueckes Peter , Bussiere Dirksen , Abrams Tinya , Rendahl Katherine , Bernard-Pierrot Isabelle , Radvanyi Francois
Published: June 2012

The mammalian fibroblast growth factor/fibroblast growth factor receptor (FGF/FGFR) family comprises 22 FGF ligands, 18 of which exert their action through 4 highly conserved transmembrane tyrosine kinase receptors (FGFR1, FGFR2, FGFR3 and FGFR4). This highly complex signaling network regulates fundamental developmental pathways, physiological processes in the adult organism and a variety of cellular functions including proliferation, differentiation, and survival. Owing to such prominent roles, it is not surprising that different types of FGF/FGFR alterations, such as abnormal expression, mutations, and amplifications, have been found in diverse tumor types. A growing body of evidence linking deregulation of the FGF/FGFR system to the pathogenesis of cancer makes these receptors attractive targets for therapeutic intervention by a small molecular weight inhibitor approach.

We have established a new and non-conventional strategy to mimic documented lead compounds which consists in the replacement of real rings by stable pseudo six-membered rings resulting from the formation of an intramolecular hydrogen bond in a planar conjugated system. More recently, we have reported an example of the application of such strategy to the design of a new class of protein kinase inhibitors featuring a N-pyrimidin-4-yl-urea motif as an alternative to the pyrido[2,3-d]pyrimidin-7-one core structure of a well-known class of protein kinase inhibitors. In this context, we have described a prototype compound displaying submicromolar activity against several tyrosine kinases including FGFR1. In this communication we illustrate the process, guided by the use of molecular modeling and biochemical and cellular assays, that led to the identification of NVP-BGJ398, a potent and selective FGFR antagonist currently in phase I clinical trial.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B246.
Abstract B26: Expression of the KLF4 and SOX10 predicting for survival in colorectal cancer.
Authors: Lee Ha-young , Rha Sun Young , Roh Jae Kyung , Chung Hyun Cheol , Hong Soojung , Park Kyu Hyun , Kim Tae Soo , Baek Eun Sil , Park Han na , Shin Sang Joon
Published: June 2012

The zinc finger transcription factor Krüppel-like factor 4 (KLF4) is involved in many important cellular processes such as growth, development, differentiation, proliferation, and apoptosis. A loss of KLF4 expression has been observed in human tumors, particularly in the gastrointestinal tract. Sry box 10 (SOX10) is a transcription factor expressed in nerve cell and melanocytes. SOX10 is believed to be essential for neural crest fate determination and to maintain the multipotency of neural crest cells. Here, to investigate the roles of KLF4 and SOX10 in colorectal cancers (CRC), we examined the expression of KLF4 and SOX10 in tumor tissues and paired normal tissues in 125 CRC patients by real-time reverse transcription-polymerase chain (real-time RT-PCR). CRC had significantly lower KLF4 and SOX10 levels compared with matched normal tissues (KLF4: 2007±1531 Copies/ul in CRC, 6585±2833 Copies/ul in normal tissues, SOX10: 60±100 Copies/ul in CRC, 399±215 Copies/ul in normal tissues) (P<0.0001). Statistically, KLF4 and SOX10 expression was not associated with age, sex, stage, lymph node metastasis, differentiation, tumor location. However, in univariate analyses, KLF4 and SOX10 were significant predictors of survival (P=0.002, P=0.039) and in multivariate analyses, KLF4 was significantly predictor of survival (P=0.045). Therefore, these findings suggest that KLF4 and SOX10 might have a potent oncogenic role in CRC tumorigenesis and prognostic value in predicting survival of CRC patients.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B26.
Abstract B32: High-throughput solution for microRNA (miRNA) isolation from stabilized whole blood in a GLP setting: Development and comparison to established procedures.
Authors: Voss Thorsten , Kruhøffer Mogens , Nielsen Karsten Bork , Vial Alexander
Published: June 2012

Introduction: Beside the analysis of messenger RNA (mRNA) expression changes, profiling of miRNAs in human whole blood holds much promise for the development of genetic markers of disease. Many scientific groups in academia and in life science companies investigate the use of miRNAs as diagnostic tools at the moment. As these small RNAs become more and more important, effective and reliable isolation procedures are urgently needed. Especially standardization and exclusion of human errors require new automated solutions which are able to replace established manual processes without losing quality and quantity of the needed analytes. In this study we compare a newly developed and optimized procedure on QIAGEN's BioRobot platform to previously published automated (Kruhøffer et al., 2007, JMD 9: 452–458) as well as manual procedures.

Material & Methods: Whole blood from consented healthy adults was collected into PAXgene Blood RNA Tubes and subjected to RNA isolation. The RNA quality and quantity were determined by using a Beckman photometer and Agilent Bioanalyzer. miRNA yields were determined by quantitative RT-PCR using commercially available probe based (Life Technologies) assays on a BioMark (Fluidigm) instrument. Due to the small reaction volume of 9 nanoliters on the BioMark arrays a pre-amplification of the cDNA was performed. Therefore 14 amplification cycles were run with a 1:100 diluted primer set which was the same that was used in the following detection assays.

Results: Compared to the originally published partly automated method the workflow could be further streamlined resulting in a fully automated procedure. With this automation the time needed for manual steps could be reduced by more than 50%. In parallel, the quality and quantity of the isolated RNA (large and small RNA species) was not affected. This is demonstrated by high RIN values (>7) and low DNA contamination levels <0.1%. With respect to miRNA enrichment, the performance of the optimized protocol was at least comparable to the current manual or partly automated methods.

Conclusion: This comparison demonstrates the high efficiency of this optimized, fully automated isolation procedure for miRNAs from stabilized whole blood. The new procedure for purification of miRNAs provides the standardization required in a GLP setting.

All miRNA enrichment protocols, the PAXgene Blood miRNA Kit and the PAXgene Blood RNA MDx Kit are for research use only. Not for use in diagnostic procedures.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B32.
Abstract B34: Receptor tyrosine kinases (RTKs) profiling and its role in advanced gastric cancer.
Authors: Hong Soojung , Ahn Jung Ryun , Zhang Xianglan , Lee Ha-young , Kim Hye Ryun , Lee Choong-kun , Lim Seungtaek , Han Jung Woo , Rha Sun Young
Published: June 2012

Background: Receptor tyrosine kinase (RTK) activation is the starting point of intracellular signaling cascades that control cellular process such as proliferation, differentiation, migration and survival and aberrant RTK activation may promote cancer initiation and progression. The purpose of this study was to determine the frequency and the influence of major RTKs including EGFR, HER-2, c-MET, and FGFR-2 expression on the outcomes of gastric cancer patients.

Methods: Between January 2000 and December 2004, 122 patients with advanced gastric cancer in our institute were selected. All patients underwent gastrectomy and D2 lymph node dissection with a curative aim and pathologically confirmed N3 stage without distant metastasis. Formalin-fixed paraffin-embedded (FFPE) tissue specimens were examined for the expression of EGFR, HER-2, c-MET, FGFR-2 by immunohistochemistry (IHC). The score for expression was assessed by the independent pathologist with weighted histoscore method. We defined positive result as the expression of 3+ in IHC for each RTK.

Results: Among 4 RTKs, c-MET expression showed highest frequency as 31 patients (25.4%) followed by FGFR-2 (13; 11.2%), HER-2 (10; 8.5%) and EGFR (6; 5.0%). The majority of samples (58.2%) were negative (wild type) for all four markers. Fourteen patients (11.5%) showed co-expression for these RTKs; one patient was quadruple positive, and the others showed double positivity for c-MET and one of 3 other markers. There were no significant correlations between each biomarker and clinic-pathologic parameters such as patient's gender, differentiation, T stage, lymphovascular invasion, Lauren classification or the number of LN metastasis. With the follow-up period of 21.5 months (0.5–115.7), median overall survival of all patients was 20.9 months (95% CI, 15.2–26.6) and median disease-free survival (DFS) was 17.1 months (95% CI, 9.4–24.8). Based on the status of each receptor, survival outcome was only associated with c-MET over-expression; median DFS of 10.8 months in c-MET positive patients and 22.0 months in c-MET negative patients (p=0.04). Median DFS in patients with co-expression of RTKs showed poorer trend than single marker positive group and wild type group (10.8m, 15.7m, and 19.4m, respectively; p=0.27).

Conclusion: We evaluated the expression pattern with four major RTKs in relatively homogenous subgroup of gastric cancer patients with pathologic N3. c-MET over-expression could be a prognostic biomarker for DFS in these patients, suggesting the significant role of c-met in gastric cancer progression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B34.
Abstract B65: Curcumin, a novel inhibitor of ATR-CHK1 pathway, suppresses homologous recombination and DNA damage checkpoint and enhances the sensitivity to PARP inhibitors.
Authors: Ogiwara Hideaki , Yokota Jun , Kohno Takashi
Published: June 2012

Curcumin is a major chemical component of turmeric (curcuma longa) and is used as a spice to give a specific flavor and yellow color in Asian foods. Here we show that treatment of (HeLa) cells with curcumin suppresses DNA damage checkpoint response and homologous recombination repair (HRR). Curcumin inhibited the phospohrylation of CHK1, but not of ATM and CHK2, induced by DNA damage. Furthermore, cells treated with curcumin failed to form ionizing radiation (IR), hydroxyurea (HU) or camptothecin (CPT)-induced RAD51 foci, but not RPA32, 53BP1 and BRCA1 foci. These results suggested that curcumin suppressed HRR and DNA damage checkpoint response through inhibition of the ATR-CHK1 pathway. In fact, curcumin led to accumulate DSBs and increase the apoptotic and necrotic cell death induced by a poly(ADP-ribose polymerase (PARP) inhibitor. It was noted that curcumin enhanced the sensitivity of cancer cells to the PARP inhibitor, which is a promising anti-cancer drug that selectively kills cells deficient for DNA damage response (DDR) including HRR and DNA damage checkpoint response. Taken together, combination of the PARP inhibitor with curcumin was indicated to have a potential to enhance the efficacy of PARP inhibitor-based cancer therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B65.