bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract B135: Secretion of R-spondin1 is regulated by C-mannosylation.
Authors: Niwa Yuki , Simizu Siro
Published: August 2014

Background and Objective: R-spondin (Rspo) family proteins (Rspo1-4) activate canonical Wnt/β-catenin signaling and noncanonical Wnt/PCP signalling, and are related to differentiation, tissue development, and sex determination. It is reported that Rspos bound to Wnt receptor (Frizzled8), Wnt co-receptor (LRP6), and LGR4-6, but the mechanism which Rspos activate Wnt signaling remains unclear.

Rspo1, a Wnt signal agonist, contains one thrombospondin type-1 repeats (TSR-1) domain. TSR-1 domain frequently involves Trp-Xaa-Xaa-Trp/Cys motif (Xaa represents any amino acids), which is the consensus sequence of protein C-mannosylation. Although several TSR-1 containing proteins have been reported to be C-mannosylated, it is not reported whether Rspo1 is C-mannosylated or not. In this study, we investigated whether Rspo1 is C-mannosylated, and the role of C-mannosylation on Rspo1 functions.

Results: We established Rspo1-overexpressing HT1080 cells, and purified recombinant Rspo1 protein from conditioned medium of the cells. Using mass spectrometric analyses, we revealed that Rspo1 is C-mannosylated at both Trp153 and Trp156. Replacement of mannosylated Trp to Ala residues inhibited secretion of Rspo1, indicating that C-mannosylation of Rspo1 is involved in the secretion. It is reported that Rspo1 synergistically activates canonical Wnt/β-catenin signaling in the presence of Wnt3a. In C-mannosylated tryptophan residues-defective Rspo1-expressing cells, Wnt3a did not activate promoter activity of TCF. Therefore, it is suggested that C-mannosylation of Rspo1 might be a new target of cancer treatment.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B135.

Citation Format: Yuki Niwa, Siro Simizu. Secretion of R-spondin1 is regulated by C-mannosylation. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B135.
Abstract B136: Study of the response of melanoma lines to BRAF/MEK inhibitors and combination drug treatments.
Authors: Dastur Anahita , Benes Cyril
Published: August 2014

The serine-threonine protein kinase, BRAF, is a frequently mutated oncogene, found in cancers of the skin, thyroid and large intestine. It is especially prevalent in malignant melanoma, where it occurs in 40-60% of the cases. The most common BRAF mutation is the valine to glutamate substitution at position 600 of the kinase (V600E). This substitution, seen in ninety percent of the BRAF cases, leads to constitutive activation of the kinase, and the downstream MEK-ERK pathway. Appropriately, several drugs have been designed to inhibit BRAF, with a broad range of outcomes in clinical trials. Unfortunately, all patients, including those showing complete responses, eventually develop resistance to single agent therapy. Our goal is to come up with effective drug combinations that would delay, or even prevent, the cancer from becoming resistant to treatment.

We used a panel of 43 melanoma lines, comprising of 28 BRAF mutants, 12 NRAS mutants and 3 lines that were wild type for these two genes. Besides BRAF and NRAS, we have the mutational status of 65 other commonly mutated cancer genes for all the lines. We assembled a set of 25 drugs targeting a wide range of proteins, including PI3K/mTOR inhibitors, the apoptosis machinery, as well as some epigenetic modulators. The melanoma lines were treated with these drugs, either (i) alone or (ii) in combination with the B-RAF inhibitor vemurafenib or (iii) in combination with the MEK inhibitor Selumetinib, for 72 hrs over a nine point, 256-fold concentration range. Viability and apoptosis were measured using cell titer glo (Promega).

The results from this pilot study are discussed here.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B136.

Citation Format: Anahita Dastur, Cyril Benes. Study of the response of melanoma lines to BRAF/MEK inhibitors and combination drug treatments. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B136.
Abstract B145: Use of μCT imaging in the PyMT breast cancer model to monitor lung metastasis development and determine therapeutic benefit in real time.
Authors: Meade Mary Anne , Balagurunathan Deepa , Bull Chris , Lister Deanne , Trachet Erin , Smith Bryan , Flynn Daniel , Wise Scott C.
Published: August 2014

Breast cancer is the most frequently diagnosed form of cancer and the second leading cause of death in women. Death and most of the complications associated with breast cancer are due to the metastasis of the primary tumor to the lung, as well as other tissues. To visualize the metastatic process in real time, the genetically engineered mouse mammary tumor virus-polyoma middle T (MMTV-PyMT) mouse model was employed along with in vivo µCT (computed tomography) imaging to evaluate and characterize the size and incidence of lung nodules. µCT images are able to detect lung nodules as small as approximately 0.5mm3. The MMTV-PyMT model is widely used to evaluate metastatic breast cancer; the model produces lung metastases (80-90% incidence rate) in a more clinically relevant manner in a syngeneic mouse with intact immune system. In this study female MMTV-PyMT mice were allowed to develop primary mammary fat pad tumors (>3grams) which were then excised and processed into single cell suspension. Syngeneic female mice (FVB/J) were implanted with the cell suspension into mammary fat pad #4 and the resulting tumors were resected once the primary tumor burden surpassed 750mg (late stage). Standard twice weekly intravenous therapy with eribulin at 1mg/kg was initiated following resection and continued the duration of the experiment. µCT images were used to assess the presence and size of lung metastases in vivo starting at 3 weeks post-primary tumor resection. Mice were monitored daily via cage side observations and imaged a second time once labored breathing was apparent. Treatment with eribulin (1mg/kg, IV) not only decreased the incidence of lung metastases by 75% compared to the vehicle control group but also inhibited the ability of the primary tumor to regrow. Coupling the MMTV-PyMT model with µCT imaging allowed for the longitudinal evaluation of therapeutic efficacy on progressive lung metastases. Visualizing lung metastases in real time with in vivo imaging is advantageous since traditional pharmacology endpoints of the MMTV-PyMT model can be extremely long (20-40 weeks).

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B145.

Citation Format: Mary Anne Meade, Deepa Balagurunathan, Chris Bull, Deanne Lister, Erin Trachet, Bryan Smith, Daniel Flynn, Scott C. Wise. Use of μCT imaging in the PyMT breast cancer model to monitor lung metastasis development and determine therapeutic benefit in real time. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B145.
Abstract B152: MR-based assessment and quantification of cerebral edema in an orthotopic mouse glioma model.
Authors: Lister Deanne R. , Balagurunathan Deepa , Baugher Meridith , Trachet Erin , McConville Patrick , Wise Scott , Leopold W.R.
Published: August 2014

Introduction: Brain edema is a prominent feature of brain cancer and contributes to neurologic dysfunction and impaired quality of life. While corticosteroids are the standard of care, improved treatments that are more efficacious and reduce side effects are sought after. In discovery of novel therapies for edema, animal brain tumor models that show relevant, reproducible and high incidence of tumor and cerebral edema are needed. Additionally, quantitative, non-invasive methods for detecting and measuring brain tumor associated edema are needed to facilitate therapeutic discovery efforts. In this work, an intracranial U251-luc human glioma model was characterized for edema incidence and progression. MRI-based relaxivity and contrast-enhancement protocols for edema detection and delineation from tumor tissue, were tested and characterized in the model.

Methods: Female nude mice were implanted intracranially with 1×10∧6 U251-luc (Luc-mCherry) glioma cells. Tumor volume assessment was performed by manual segmentation of T1-weighted, gadolinium-enhanced anatomical brain images based on the assumption of tumor enhancement due to leaky vessels or compromised blood-brain barrier. Edema assessment was performed by manual segmentation of T2-weighted anatomical brain images based on the assumption that enhancing regions include tumor tissue in addition to regions of brain edema. The difference in T2-weighted and T1-weighted volumes was assumed to be edema. T2 maps were also generated over the whole brain to measure T2 values over the respective regions of interest, and confirm the differentiation between tumor tissue and edema.

Results: Edema volume was successfully distinguished from contrast enhancing tumor tissue and quantified in orthotopically implanted U251-luc (Luc-mCherry) glioma. Incidence was 100%. Edema progression occurred with increasing tumor volume over time. Regions of edema exhibited greater T2 values compared to tumor tissue regions (that also had greater T2 than normal tissue). This is consistent with edematous tissue water content, compared with tumor tissue and normal brain. These results support the use of intracranial U251-luc (Luc-mCherry) as a reliable model for studying the effects of therapies targeting tumor-related cerebral edema, and the application of MR imaging for quantifying those effects real-time in vivo.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B152.

Citation Format: Deanne R. Lister, Deepa Balagurunathan, Meridith Baugher, Erin Trachet, Patrick McConville, Scott Wise, W.R. Leopold. MR-based assessment and quantification of cerebral edema in an orthotopic mouse glioma model. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B152.
Abstract B159: Heterozygous IDH1 mutations modify the citric acid (TCA) cycle metabolism and sensitize cells to inhibition of mitochondrial respiration/oxidative phosphorylation.
Authors: Grassian Alexandra R. , Parker Seth , Davidson Shawn , Green Courtney , Lin Fallon , Joud-Caldwell Carol , Yin Hong , Chung Franklin , Straub Christopher , Vander Heiden Matthew , Pagliarini Raymond , Metallo Christian
Published: August 2014

Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) occur in a variety of tumor types. Although these mutations are loss-of-function for conversion of isocitrate to α-ketoglutarate, the mutant enzymes greatly increase the production of the proposed oncometabolite, 2-hydroxyglutarate (2-HG). However the full metabolic consequences of IDH1/2 mutation in their heterozygous cellular context have yet to be fully explored. To address this question, we utilized a panel of isogenic cell lines with wild-type IDH1/2 or clinically relevant IDH1/2 mutations and examined the metabolic consequences of IDH mutation using (13)C metabolic flux analysis (MFA).

We observe a dramatic and consistent decrease in the ability of IDH1 mutant cell lines to utilize reductive glutamine metabolism via the carboxylation of α-ketoglutarate back to isocitrate. This was not seen either in IDH2 mutant cell lines or in wild-type cell lines treated with exogenous 2-HG. Consistent with these changes, the IDH1 mutant cell lines, but not IDH2 mutant or 2-HG treated cells, were deficient in the utilization of glutamine for de novo lipogenesis. Similar trends were observed in endogenous, non-engineered IDH1/2 mutant cell lines.

The decrease in reductive carboxylation in the IDH1 mutant cell lines raises the hypothesis that these cells may be more reliant on mitochondrial metabolism. Indeed, IDH1 mutant cells were more sensitive to either treatment with an electron transport chain inhibitor or growth in hypoxia (which also inhibits mitochondrial metabolism).

These results show heterozygous IDH1 mutation robustly impacts wild-type cellular metabolism in a different manner than IDH2 mutation. Furthermore, these results suggest that IDH1 and IDH2 mutant tumors may be differentially sensitive to inhibitors of specific metabolic pathways.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B159.

Citation Format: Alexandra R. Grassian, Seth Parker, Shawn Davidson, Courtney Green, Fallon Lin, Carol Joud-Caldwell, Hong Yin, Franklin Chung, Christopher Straub, Matthew Vander Heiden, Raymond Pagliarini, Christian Metallo. Heterozygous IDH1 mutations modify the citric acid (TCA) cycle metabolism and sensitize cells to inhibition of mitochondrial respiration/oxidative phosphorylation. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B159.
Abstract B169: Hypoxia-inducible factor 1α (HIF-1α) correlates with epithelial-mesenchymal transition (EMT) and tumor growth in thyroid cancer cell lines.
Authors: Yang YJ , Na HJ , Kim Ws , Bong Jp , Baek Sj , Kim Jw , Park Jh , Shin Dy , Hong Hj , Kwon Jh , Lee Ej , Koh Yw , Choi Ec
Published: August 2014

Hypoxia is essential for microenvironment to induce tumor metastasis. Intratumoural hypoxia, followed by stabilization and activation of the HIF-1α transcription complex, is one of the most important mechanisms that promotes metastasis and thereby, increases tumor aggressiveness. EMT (epithelial to mesenchymal transition), one of the central mechanisms that induces invasion and metastasis of tumors. The aim of this study was to investigate the possible role of HIF-1α as one of the microenvironmental influences driving metastatic progression by induction of epithelial to mesenchymal transition in thyroid cancer. In vitro studies revealed a functionally active HIF-1a pathway in several types of thyroid cells with transcriptional activation observed after hypoxia or overexpression. Knock-down of HIF-1α or overexpression of HIF-1α in thyroid cancer cells suggested that HIF-1α promotes epithelial-mesenchymal transition (EMT) and metastastic phenotypes in follicular thyroid cancer cell lines, especially. HIF-1α increases the expression of Vimentin, N-cadherin, Snail, Slug, Twist, MMP2, VEGF but not E-cadherin. Also, wound healing assay and in vitro migration assays showed that the cell migration ability was increased in a HIF1α- dependent manner. Furthermore, knock-down of HIF-1α leads to decreased tumor growth and regulates the expression of EMT marker genes in thyroid tumor xenograft mouse model. Taken together, these results suggest that the HIF-1 signaling induces EMT and metastasis through the regulation of various EMT regulators in vitro as well as in vivo study.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B169.

Citation Format: YJ Yang, HJ Na, Ws Kim, Jp Bong, Sj Baek, Jw Kim, Jh Park, Dy Shin, Hj Hong, Jh Kwon, Ej Lee, Yw Koh, Ec Choi. Hypoxia-inducible factor 1α (HIF-1α) correlates with epithelial-mesenchymal transition (EMT) and tumor growth in thyroid cancer cell lines. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B169.
Abstract B190: GDC-0068 is a novel and selective Akt inhibitor that enhances the efficacy of FOLFOX in primary gastric cancer models.
Authors: Ingalla Ellen , Hong Rebecca , Savage Heidi , Punnoose Elizabeth , Rost Sandra , Koeppen Hartmut , Sampath Deepak , Nannini Michelle A.
Published: August 2014

Background: Akt, a serine/threonine protein kinase, is a key signaling node in the PI3K/Akt/mTOR pathway and plays an essential role in regulating tumor cell proliferation, migration and survival. PI3K/Akt signaling is up-regulated in gastric cancer, mainly due to loss of PTEN, and is associated with poor prognosis and chemoresistance. Chemotherapeutics such as doxorubicin and 5-FU/cisplatin have been shown to upregulate PI3K/Akt signaling in several solid cancers in response to DNA damage. We have previously reported on a potent highly selective ATP-competitive pan-Akt inhibitor, GDC-0068, which inhibits cell cycle progression and viability of cancer cell lines and xenografts driven by Akt signaling, including those with defects in the tumor suppressor PTEN, oncogenic mutations in PIK3CA, and amplification of HER2. Thus, we hypothesized that the combination of FOLFOX and GDC-0068 would induce anti-tumor activity in primary gastric cancer models.

Methods: Gastric cancer cell lines (n=11) were treated with increasing concentrations of GDC-0068 and FOLFOX and evaluated for combinational efficacy over single agent via cell viability assay. For in vivo studies, primary gastric cancer xenograft models were implanted in immuno-compromised mice and treated with GDC-0068 and FOLFOX as single agents or in combination.

Results: In vitro, the combination of GDC-0068 and FOLFOX results in enhanced inhibition of tumor cell viability in the cell lines with activation of the PI3K pathway compared to either single agent alone. These results were recapitulated in vivo, in the primary xenograft models STO#240 and STO#182, where the combination of GDC-0068 and FOLFOX resulted in increased tumor growth inhibition or regressions compared to single agents alone. All combinations tested were well tolerated in vivo based on minimal changes in body weights.

Conclusions: Our preclinical studies demonstrate improved anti-tumor efficacy when GDC-0068 is combined with FOLFOX in gastric cancer models in vitro and in vivo. The data supports the clinical development of GDC-0068 in combination with FOLFOX for the treatment of gastric cancer.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B190.

Citation Format: Ellen Ingalla, Rebecca Hong, Heidi Savage, Elizabeth Punnoose, Sandra Rost, Hartmut Koeppen, Deepak Sampath, Michelle A. Nannini. GDC-0068 is a novel and selective Akt inhibitor that enhances the efficacy of FOLFOX in primary gastric cancer models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B190.
Abstract B221: Hmga2 furthers lung adenocarcinoma progression through function as a competing endogenous RNA.
Authors: Kumar Madhu S. , Armenteros-Monterroso Elena , East Philip , Chakravorty Probir , Matthews Nik , Winslow Monte M. , Downward Julian
Published: August 2014

Lung adenocarcinoma is the most widespread cancer type worldwide. As the majority of patients are diagnosed with metastatic disease, it is vital to understand that biological basis of adenocarcinoma progression. Hmga2 is among the most over-expressed genes in lung adenocarcinoma, and we have previously shown it is necessary for lung cancer progression in vivo. However, the mechanism of Hmga2-mediated cancer progression is unclear. Here we demonstrate Hmga2 promotes oncogenic transformation by functioning as a competing endogenous RNA (ceRNA) for the let-7 family of microRNAs (miRNAs). First, Hmga2 can promote lung cancer cell transformation independent of its protein-coding function but dependent upon let-7 sites in its 3’ untranslated region. These effects are observed both in vitro and in vivo, where Hmga2 ceRNA activity promotes lung cancer growth and dissemination, impairing survival. Combined analyses of miRNA target prediction and cell line gene expression data suggested the TGF-β co-receptor Tgfbr3 lies downstream of the Hmga2 ceRNA. Follow-up studies demonstrated Hmga2 specifically regulated Tgfbr3 expression through differential recruitment of the miRNA regulatory machinery. This regulation is functionally relevant, as both Tgfbr3 in particular and TGF-β in general are necessary for Hmga2 to promote transformation. To assess the clinical relevance of these experimental findings, we assessed gene expression data from two extensive lung cancer patient gene expression datasets: the Cancer Genome Atlas and the Director's Challenge. Analysis of this gene expression data revealed that HMGA2 and TGFBR3 are coordinately and reciprocally regulated in patients, a necessary prediction of HMGA2 ceRNA function. In total, these results indicate the Hmga2 oncogene functions as both a protein-coding gene and a ceRNA. Such multi-level regulation of gene expression constitutes a novel paradigm by which genes alter cancer development and progression through combined function as both coding proteins and non-coding RNAs.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B221.

Citation Format: Madhu S. Kumar, Elena Armenteros-Monterroso, Philip East, Probir Chakravorty, Nik Matthews, Monte M. Winslow, Julian Downward. Hmga2 furthers lung adenocarcinoma progression through function as a competing endogenous RNA. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B221.
Abstract B23: Apoptotic and autophagic effects of Sesbania grandiflora flowers in human leukemic cells.
Authors: Roy Rajneeta , Kumar Deepak , Chowdhury Chinmay , Das Padma
Published: August 2014

Background: Identification of cytotoxic compounds that induce apoptosis has been the mainstay of anticancer therapeutics for several decades. In recent years, focus has shifted to inducing multiple modes of cell death coupled with reduced systemic toxicity. The plant Sesbania grandiflora is widely used in Indian traditional medicine for the treatment of a broad spectrum of diseases. This encouraged us to investigate into the anti-proliferative effect of a fraction (F2) isolated from S. grandiflora flowers in cancer cells and delineate the underlying involvement of apoptotic and autophagic pathways.

Principal Findings: Using MTT based cell viability assay, we evaluated the cytotoxic potential of fraction F2. It was the most effective on U937 cells (IC50: 18.6 µg/ml). Inhibition of growth involved enhancement of Annexin V positivity. This was associated with elevated reactive oxygen species generation, measured by flow cytometry and reduced oxygen consumption– both effects being abrogated by antioxidant NAC. This caused stimulation of pro-apoptotic proteins and concomitant inhibition of anti-apoptotic protein expressions inducing mitochondrial depolarization, as measured by flow cytometry and release of cytochrome c. Interestingly, even with these molecular features of apoptosis, F2 was able to alter Atg protein levels and induce LC3 processing. This was accompanied by formation of autophagic vacuoles as revealed by fluorescence and transmission electron microscopy– confirming the occurrence of autophagy. Eventually, F2 triggered caspase cascade– executioners of programmed cell death and AIF translocation to nuclei. This culminated in cleavage of the DNA repair enzyme, poly (ADP-ribose) polymerase that caused DNA damage as proved by staining with Hoechst 33258 leading to cell death.

Conclusions: The findings suggest fraction F2 triggers pro-oxidant activity and mediates its cytotoxicity in leukemic cells via apoptosis and autophagy. Thus, it merits consideration and further investigation as a therapeutic option for the treatment of leukemia.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B23.

Citation Format: Rajneeta Roy, Deepak Kumar, Chinmay Chowdhury, Padma Das. Apoptotic and autophagic effects of Sesbania grandiflora flowers in human leukemic cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B23.
Abstract B242: Adipocytes contribute to resistance of human melanoma cells to chemotherapy and targeted therapy.
Authors: Chi Mengna , Chen Jiezhong , Ye Yan , Tseng Hsin-Yi , Lai Fritz , Tay Kwang Hong , Jin Lei , Jiang Chen Chen , Zhang Xu Dong
Published: August 2014

Increasing epidemiological evidence has linked the development and progression of several cancers including melanoma with obesity. However, whether obesity impinges on responses of cancer cells to treatment remains less understood. Here we report that human adipocytes contribute to resistance of melanoma cells to various therapeutic agents. Exposure to media from adipocyte cultures (adipocyte media) increased cell proliferation and reduced sensitivity of melanoma cells to apoptosis induced by diverse chemotherapeutic drugs, including the DNA-damaging drug cisplatin, the microtubule-targeting agent docetaxel, and the histone deacetylase inhibitor SAHA. This was associated with increased activation of PI3K/Akt and MEK/ERK signaling, and was attenuated by a PI3K or MEK inhibitor. The effect of adipocyte media on melanoma cells was, at least in part, due to the interaction between the adipokine leptin and its long form receptor OB-Ob, in that immunodepletion of leptin in adipocyte media or siRNA knockdown of OB-Rb in melanoma cells reversed the increase in Akt and ERK activation, enhancement in cell proliferation, and importantly, protection of melanoma cells against the drugs. In support, recombinant leptin partially recapitulated the effect of adipocyte media on melanoma cells. Of note, OB-Rb was increased on the surface of melanoma cells compared to melanocytes, whereas leptin short form receptors appeared to be suppressed post-transcriptionally, suggesting that OB-Rb was selectively upregulated in melanoma cells. Collectively, these results indicate that adipocytes contribute to resistance of melanoma cells to chemotherapeutic drugs and agents targeting the PI3K/Akt and MEK/ERK pathways, and suggest that inhibition of the leptin/ OB-Rb system may be useful to improve the efficacy of multiple therapeutic approaches in the treatment of melanoma.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B242.

Citation Format: Mengna Chi, Jiezhong Chen, Yan Ye, Hsin-Yi Tseng, Fritz Lai, Kwang Hong Tay, Lei Jin, Chen Chen Jiang, Xu Dong Zhang. Adipocytes contribute to resistance of human melanoma cells to chemotherapy and targeted therapy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B242.
Abstract B280: Histone deacetylase (HDAC) 6 inhibition potentiates the cytotoxicity of lapatinib and PI3K inhibitors on Her2+ breast cancer cell lines.
Authors: Yang Min , Tamang David , Jones Simon
Published: August 2014

ACY-1215 is a novel and selective oral inhibitor of HDAC6 currently in Phase I clinical trials in combination with bortezomib or lenalidomide in relapsed/refractory multiple myeloma. Inhibitors of HDAC6 have demonstrated anti-cancer proliferative activity in various cancer types, including Her2+ breast cancer. Blocking HDAC6 activity has been shown to cause Her2+ breast cancer cell growth inhibition through various mechanisms, including destabilization of Her2 mRNA and protein. In the present work, the impact of HDAC6 inhibition has been investigated on Her2+ breast cancer cell growth and cell migration using HDAC6 inhibitor alone or in combination with either a Her2 inhibitor (lapatinib) or a PI3K inhibitor (GDC-0941, IPI-145 and CAL-101).

Anti-tumor activity of ACY-1215 was investigated using a panel of nearly 100 human tumor cell lines derived from the major cancer types and Her2+ breast cancer cells were among the most sensitive cells to ACY-1215. Combinations of ACY-1215 with either the Her2 inhibitor lapatinib or PI3K inhibitor GDC-0941 demonstrated synergistic cytotoxicity against these cancer cells. To illustrate the effects are due to HDAC6 inhibition, we used a highly HDAC6 selective inhibitor ACY-775, which has greater than 1,500 fold of selectivity against HDAC6 over Class I HDACs (HDAC1, 2 and 3), in our experiments. ACY-775 is able to sensitize Her2+ breast cancer cells MDA-MB-453 and BT-474 to lapatinib, and PI3K inhibitors (IPI-145, GDC-0941 and CAL-101). ACY-775 also blocks cancer cell migration in the presence of absence of epidermal growth factor (EGF). In agreement with the proposed mechanism of HDAC6 mediated Her2+ breast cancer cell death, ACY-1215 treatment correlated with decrease of Her2 protein, p-EGFR and p-AKT in a Her2+ breast cancer cell line MDA-MB-453.

By taking advantage of our highly selective HDAC6 inhibitor ACY-775, we have shown growth and migration suppressive activities by HDAC6 inhibition in Her2+ breast cancer cells. These findings suggest selection of metastatic Her2+ breast cancer for further in vitro and in vivo validation and mechanism of action studies with ACY-1215 in combination with novel and approved standard of care therapeutics for breast cancer.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B280.

Citation Format: Min Yang, David Tamang, Simon Jones. Histone deacetylase (HDAC) 6 inhibition potentiates the cytotoxicity of lapatinib and PI3K inhibitors on Her2+ breast cancer cell lines. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B280.
Abstract B285: A novel, potent, highly selective inhibitor of c-Met kinase, Simm559, inhibits c-Met-dependent neoplastic phenotypes in vitro and in vivo.
Authors: Ai Jing , Zhan ZhengSheng , Wang Ying , Chen Fang , Peng Xia , Chen Yi , Ji Yinchun , Ding Jian , Geng Meiyu , Duan Wenhu
Published: August 2014

Despite the vigorous activity in the development of c-Met inhibitors, none of the c-Met inhibitors or c-Met pathway antagonists has been approved for clinical use. Notably, most of the reported c-Met kinase inhibitors across the clinical trials lack selectivity and target multiple kinases, which will likely result in unwanted off-target toxicities. The discovery of highly selective c-Met inhibitors may represent an important direction to increase chances of targeting c-Met for cancer therapy. Simm559 is identified as a highly selective and potent c-Met inhibitor, with IC50 = 0.24 ± 0.04 nM against c-Met kinase, while it barely inhibited kinase activity of a panel of tyrosine kinases, including c-Met family member and its highly homologous kinases. Simm559 significantly inhibited c-Met-driven cell proliferation, in particular c-Met constitutively activated NSCLC and gastric cancer cells, with an average IC50 at 0.66 nM. Such potency was over 10,000-fold of that in cells with low c-Met expression or activity. Oral dosing of Simm559 at 25 mg/kg (BID) resulted in significant antitumor activity in several c-Met-driven xenograft models, especially in NSCLC EBC-1 xenograft, gastric SNU5 xenograft and a patient-derived NSCLC xenograft model with MET amplification. Complete tumor regressions were observed in both cell line-based and patient-derived lung cancer xenograft models. Safety evaluation and pharmacokinetic analysis showed that Simm559 is safe and well tolerant with acceptable pharmacodynamic properties. All these evidence promise Simm559 as a therapeutic candidate for c-Met-driven human cancers.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B285.

Citation Format: Jing Ai, ZhengSheng Zhan, Ying Wang, Fang Chen, Xia Peng, Yi Chen, Yinchun Ji, Jian Ding, Meiyu Geng, Wenhu Duan. A novel, potent, highly selective inhibitor of c-Met kinase, Simm559, inhibits c-Met-dependent neoplastic phenotypes in vitro and in vivo. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B285.
Abstract B288: 17AAG enhances cisplatin-induced cytotoxicity to induce p53-mediated apoptosis in head and neck cancer.
Authors: Kwon Minsu , Kim Eun Hye , Park Hyun Bae , Kim Sang Yoon , Roh Jong-Lyel
Published: August 2014

Purpose: Tumor suppressor p53 is often inactivated in head and neck cancer (HNC) because of TP53 mutations or the overexpression of MDM2 or MDMX. Restroation of p53 function through counteracting these p53 repressors is considered a promising strategy for cancer treatment. Therefore, we examined the role of a heat shock protein 90 inhibitor, 17-(Allylamino)-17-demethoxygeldanamycin (17AAG), in inducing apoptosis in HNC by restoring p53 function.

Experimental Design: In five HNC cell lines, we tested the effects of 17AAG treatment, either individually or in combination with 17AAG or cisplatin and assessed growth suppression, cell cycle arrest, and apoptosis assay.

Results: 17AAG activated and stabilized p53 in an HNC cell line bearing wild-type TP53 by disrupting the p53-MDMX interaction. 17AAG upregulated expression of p21 and proapoptotic genes and promoted apoptosis, in a dose-dependent manner. 17AAG exerted the highest growth suppression in tumor cells with MDMX overexpression. The apoptotic response was blocked by inhibition of p53 or MDMX expression, demonstrating that the effects of 17AAG depend on p53 and MDMX. In combination treatments, 17AAG acted synergistically with Nutlin-3a to inhibit growth of HNC cells. 17AAG also synergized the antitumor effect of cisplatin by inducing p53-mediated apoptosis.

Conclusion: 17AAG effectively induced p53-mediated apoptosis in HNC cells by MDMX inhibition, suggesting a promising strategy for treating HNC.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B288.

Citation Format: Minsu Kwon, Eun Hye Kim, Hyun Bae Park, Sang Yoon Kim, Jong-Lyel Roh. 17AAG enhances cisplatin-induced cytotoxicity to induce p53-mediated apoptosis in head and neck cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B288.
Abstract B289: Inhibition of platelet-derived growth factor receptors by ARQ 087, a multi-tyrosine kinase inhibitor.
Authors: Yu Yi , Lee Jennifer , Dransfield Daniel T.
Published: August 2014

Background: The platelet-derived growth factor (PDGF) receptors are receptor tyrosine kinases which contain two isoforms, PDGFRα and β. They form homo- or hetero-dimers and exert different biological functions including organ development and vascularization. Dysregulation of PDGF receptors has been implicated in tumor initiation and progression in addition to playing a major role in epithelial-mesenchymal transition, metastasis, and tumor angiogenesis. ARQ 087, a pan FGF receptor inhibitor inhibits FGFR1, 2, and 3 with IC50 from 2 to 4 nM.

Methods: We have performed in vitro kinase profiling to determine percent inhibition of selected kinases and IC50 for PDGFRα and its mutated form. siRNA knockdown was applied to evaluate cell proliferation of the PDGFRα-dependent cancer cell lines NCI-H1703 and A-204. Western blot analysis including phospho PDGFRα/β, pSTAT3, pAKT, pERK together with cell migration and invasion analysis was performed on NCI-H1703 and NIH-3T3 cells.

Results: ARQ 087 is an inhibitor of PDGF receptors which is supported by the in vitro kinase profiling results. ARQ 087 but not Imatinib, was equally potent against wild-type PDGFRα and the mutated form of PDGFRα (D842V), indicating a potential clinical implication for Imatinib-resistant PDGFR-driven cancers. Knockdown of PDGFRα using siRNA suppressed PDGFRα-dependent cancer cell line proliferative activity. ARQ 087 inhibition of cell proliferation was accompanied by the reduction of phosphorylation of PDGFRα and its downstream proteins. In addition to the anti-proliferative activity, ARQ 087 suppressed migration and invasion of NCI-H1703 cells through targeting PDGFRα and migration of NIH-3T3 cells through targeting PDGFRβ.

Conclusions: ARQ 087 is not only an inhibitor of the FGFR family but also of the PDGFR family, suggesting that targeting cancers driven by PDGF receptors or mutated forms may provide an additional clinical path for ARQ 087.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B289.

Citation Format: Yi Yu, Jennifer Lee, Daniel T. Dransfield. Inhibition of platelet-derived growth factor receptors by ARQ 087, a multi-tyrosine kinase inhibitor. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B289.
Abstract B300: Discovering immune-mediated therapy for cancer biologics is informed by in vitro cellular assays.
Authors: Williams Geoff , Poon Edmund , Ireland Samantha , Dillon Janette , Stewart Ross , Wilkinson Robert
Published: August 2014

Immune-mediated therapy for cancer (IMT-C) biologics are an emerging class of cancer drugs which target cells of the immune system to enhance the immune response to cancer. Cross-reactivity of IMT-C mAbs and other biologics to rodents is rare, emphasizing the importance of human in vitro assays in IMT-C discovery and development. Typically there is a trade-off between the physiological relevance of an assay and its throughput, hence higher-throughput assays are used in the early stages of drug discovery, and more complex, lower-throughput assays are subsequently used to profile lead candidates. Here, we review a panel of in vitro cellular assays currently used for IMT-C drug programs at MedImmune including transgenic reporter cell systems, bead-based assays, and multi-cellular systems using cell-lines and primary human immune cells.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B300.

Citation Format: Geoff Williams, Edmund Poon, Samantha Ireland, Janette Dillon, Ross Stewart, Robert Wilkinson. Discovering immune-mediated therapy for cancer biologics is informed by in vitro cellular assays. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B300.
Abstract B34: Development of a urine microvesicle/exosome RNA biomarker panel to identify prostate cancer.
Authors: Skog Johan , Noerholm Mikkel , Bentink Stefan , Romain Charlotte , Fishbeck Jillian , Sinclair Ian , Scott Anna , Mueller Romy , Koestler Tina , Belzer Susan
Published: August 2014

Introduction: Prostate cancer is diagnosed utilizing anywhere from 10-12 transrectal prostate needle biopsy specimens (PNB). The decision to biopsy is driven in part by the PSA test, which is known to have marginal accuracy in appropriate patient selection. As a result, between 40 to 60% of patients will have a benign biopsy with approximately 20% having false negative results. Exosomal RNA is stable in urine and can be easily extracted from a random urine sample. A novel molecular assay with an improved ability to predict the likelihood of having prostate cancer on a needle biopsy is being developed through isolation of RNA from urine exosomes and subsequent identification of an accurate prognostic gene signature.

Methods: A multicenter study was designed to collect first catch random urine, without a prior digital rectal exam (DRE) from men presenting for a PNB. Urine samples were processed at Exosome Diagnostic's clinical laboratory under quality control using the EXO70 Urine Microvesicle RNA Isolation Kit. The exosomal RNA was reverse transcribed and qPCR assays for specific genes performed. The results of those assays along with collected clinical data and PNB histopathology were subjected to statistical analysis to derive ROC curves.

Results: Urine samples from 258 men scheduled for a PNB were tested. Triaged gene lists previously shown to be important in prostate cancer were compiled and univariately assessed with respect to discrimination of PNB results. A series of single and multiple gene expression profiles representing anywhere from 1 to 3 gene signatures relative to a reference gene were identified. Multivariate models including several gene signatures with a ROC AUC performance > 0.7 were found. Interestingly, a 3 gene signature composed of PCA3, ERG and KLK3 provided a very good performance with an AUC of 0.76 (N = 122).

Conclusions: Prostate cancer cells release exosomes / microvesicles that can be isolated from a random urine sample. The exosomal RNA is stable and can be efficiently isolated and used for tumor profiling even without a prior DRE. This urine exosome platform presents a robust model for a quick and simple test that can assist in the initial biopsy decision process.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B34.

Citation Format: Johan Skog, Mikkel Noerholm, Stefan Bentink, Charlotte Romain, Jillian Fishbeck, Ian Sinclair, Anna Scott, Romy Mueller, Tina Koestler, Susan Belzer. Development of a urine microvesicle/exosome RNA biomarker panel to identify prostate cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B34.
Abstract B36: Expression of growth and cell proliferation factors in gastric carcinomas: The association with poor overall survival on patients with intestinal type.
Authors: Begnami Maria D. , Pinto Clovis AL , Nonogaki Sueli , Soares Fernando , Lima Luiz GCA
Published: August 2014

Background: Gastric carcinomas (GC) express a variety of growth factors and cell proliferation acting in the mechanisms of tumor invasion and spread. The expression of angiogenic factors such as vascular endothelial growth factor (VEGF) has been demonstrated in diffuse type GC, whereas expression of growth factors is associated with intestinal-type carcinomas. Objective: To study the immunohistochemical expression of proteins associated with growth factors and cell proliferation in GC. Methods: We studied 400 GC, arranged in duplicates in 2 blocks of tissue microarray (TMA). Immunohistochemistry was performed using antibodies: c-MET (Novocastra), TGFbetaI (Santa Cruz), TGFbetaII (Santa Cruz), c-erbB-2 (Dako) and VEGF (Santa Cruz). Cases were considered positive for TGFbetaI, TGFbetaII and VEGF when cytoplasmic staining was observed in more than 10% of tumor cells. Positive cases for c-met and c-erbB-2 were detected when strong staining on the membrane cells was observed in more than 10% of neoplastic cells. Results: Expression of TGFbetaI, TGFbetaII and VEGF were detected in 314/385 (81%), 370/382 (96%) and 333/366 (90%) of GC. 333/376 (88%) cases were positive for c-met and 54/385 (14%) for c-erbB-2. Intestinal type carcinomas were more often positive for TGFbetaII (p = 0.01), VEGF (p = 0.001), c-met (p = 0.01), and c-erbB-2 (p = 0.001). According to univariate statistical analyzes expressions of TGFbeta II, c-met and c-erbB-2 were independent factors associated with overall survival of patients with GC. Conclusions: The proteins associated with growth factors and cell proliferations are differentially expressed in intestinal type of GC. Our findings pointed them out as biological factors associated with worse prognosis and important therapeutic targets.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B36.

Citation Format: Maria D. Begnami, Clovis AL Pinto, Sueli Nonogaki, Fernando Soares, Luiz GCA Lima. Expression of growth and cell proliferation factors in gastric carcinomas: The association with poor overall survival on patients with intestinal type. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B36.
Abstract B44: Assessing factors predictive of response to ADCs for companion diagnostic strategies.
Authors: Krueger Joseph S. , Lange Holger , Potts Steve , Young David
Published: August 2014

One premise of antibody-drug conjugates (ADC) is that the bound mAb-antigen complex on the cell surface will internalize and be metabolized by lysosomal proteases to release the free drug. Thus, the efficacy of an ADC is dependent not only on the presence of cell surface antigens, but also in-tact delivery of the conjugated drug, and an active pathway of receptor-mediated endocytosis. On a cellular level, the biological mechanisms behind receptor internalization and turnover have not been elucidated for novel therapeutic targets. On a tissue level, vascularization and hypoxic profile will affect delivery of the antibody into the tissue. Varying expression of the antibody target within the tissue (antigen density) will also affect the uptake of the ADC. Furthermore, the extracellular stability of the ADC may be affected by the activity of the various proteases in the tumor microenvironment (TME), outside of the lysozome. These concepts are critical for efficacy, and thus are especially important for companion diagnostic approaches (CDx) meant to predict response to ADCs. Thus, a predictive assay which would account not only for the degree of cell surface expression of the target, but also receptor internalization and potential effects of tumor microenvironment is required. To answer this need, Flagship Biosciences has invented several proprietary approaches for measuring critical properties of the therapeutic target on the cell surface or inside the cell, as well as properties of the TME which could be used to understand and predict efficacy to an ADC using FFPE biopsies. These quantitative pathology approaches are based on image analysis approaches which been designed specifically for ADC CDx programs to answer these critical questions: 1) Defining cell surface target expression independent of cytoplasmic expression; 2)Estimating receptor flux (turnover, internalization) based on staining profile; 3) Assessing critical factors in the TME which may affect delivery of the in-tact drug to the intracellular target; 4) Assaying vascular properties which may affect delivery of the drug; and 5)Heterogeneity of the target within a tumor. We are able to provide these as discrete evaluations or multiplex these evaluations for an integrative answer which can be derived from a typical clinical biopsy. Incorporation of these novel tools will enable ADC developers to create efficacy and patient stratification paradigms which incorporate the critical biological endpoints unique to ADCs.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B44.

Citation Format: Joseph S. Krueger, Holger Lange, Steve Potts, David Young. Assessing factors predictive of response to ADCs for companion diagnostic strategies. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B44.