bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 912: Targeting HER family signaling in low HER2-expressing breast cancer: activity of the selective and equipotent EGFR, HER2 and HER3 signaling inhibitor, AZD8931, in models of low HER2-expressing disease.
Authors: Marshall Gayle , Ashton Susan , Speake Georgina , D'Cruz Celina , Grondine Michael , Trigwell Cath , Bigley Graham , Beran Garry , Lynaugh Katy , Klinowska Teresa C.
Published: August 2014

Effective therapies for women with low-HER2 expressing breast cancer remain a significant unmet clinical need. We have detected significant phosphorylated-HER and HER2:HER3 dimer expression in clinical breast cancer samples without HER2 amplification suggesting that HER signaling may play a role in these tumors. It is our hypothesis that ligand-driven signaling is the major route of HER family activation in the absence of HER2 amplification.

AZD8931 is an orally bioavailable and highly selective small molecule inhibitor of EGFR, HER2 and HER3 signaling, which shows greatest potency when HER signaling is ligand-driven(1). In a range of low HER2 breast cancer cell lines, AZD8931 inhibited heregulin-driven proliferation (GI50 range 0.05 to 0.38μM) HER2:HER3 signaling, HER3:PI3K interaction and downstream signaling. In vivo, twice-daily oral dosing of AZD8931 showed significant monotherapy anti-tumor efficacy in a xenograft model of low HER2 breast cancer (MDA-MB-175VII) at well-tolerated doses (90% TGI at 12.5 mg/kg/bid).

We further evaluated the activity of AZD8931 in combination with paclitaxel, a commonly used standard of care chemotherapy for patients with advanced breast cancer expressing low levels of HER2. Pre-clinically, the combination of AZD8931 with paclitaxel showed at least additive activity in vitro in a range of breast cell lines and additive efficacy in vivo in a BT474 (high HER2) xenograft model at well tolerated doses (AZD8931 12.5 mg/kg/bid TGI 63%; paclitaxel 7.5mg/kg/qw TGI 41%; combination TGI 95%). Combination work in the low HER2 xenograft model is ongoing.

These data demonstrate that AZD8931 inhibits ligand-driven HER family receptor activation, receptor dimerization, PI3K interaction and downstream signaling leading to anti-tumor activity in vivo. These data also support the potential clinical utility of AZD8931 for the therapeutic treatment of low HER2 expressing breast cancers in combination with paclitaxel.

1 Hickinson et al. Clin.Cancer Res (2010) 16:1159-69.

Citation Format: Gayle Marshall, Susan Ashton, Georgina Speake, Celina D'Cruz, Michael Grondine, Cath Trigwell, Graham Bigley, Garry Beran, Katy Lynaugh, Teresa C. Klinowska. Targeting HER family signaling in low HER2-expressing breast cancer: activity of the selective and equipotent EGFR, HER2 and HER3 signaling inhibitor, AZD8931, in models of low HER2-expressing disease. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 912. doi:10.1158/1538-7445.AM2013-912
Abstract 92: Comparative expression analysis of somatostatin receptor subtypes in human neuroendocrine tumors using two sets of new monoclonal antibodies.
Authors: Lambertini Chiara , Barzaghi-Rinaudo Patrizia , D'Amato Lisa , Schulz Stefan , Nuciforo Paolo , Schmid Herbert A.
Published: August 2014

The expression and reliable detection of somatostatin receptor subtypes (SSTR1-5) is a prerequisite for the successful use of somatostatin analogues in neuroendocrine tumors (NET). Two sets of monoclonal antibodies (mAbs) against human SSTR1, 2A, 3 and 5 have recently been developed by two independent laboratories using rabbit and mouse hybridomas. Our aim was to evaluate the usefulness of both sets of mAbs for detection of SSTRs in NET samples as they are routinely collected in clinical practice. Mouse and rabbit mAbs were characterized in SSTR1, 2, 3 and 5-transfected HEK293 cells and human archival pancreatic tissue and NET samples. Comparative analysis of mAbs was also conducted by immunostaining of a tissue microarray composed of 75 cores of NET. Immunohistochemical analysis of HEK293 cells showed that both rabbit and mouse mAbs specifically detect their cognate receptor subtype, with mild cytoplasmic cross-reactivity observed for rabbit mAbs. Both sets of mAbs labeled normal pancreatic islets and showed similar patterns of immunoreactivity in NET controls. Direct comparison of mAb sets using a NET tissue microarray revealed a strong correlation between rabbit and mouse mAbs against SSTR1 and 5, and moderate correlation for SSTR3. The rabbit mAb against SSTR2A showed higher affinity for its cognate receptor than the corresponding mouse mAb, resulting in more reliable detection of this SSTR. In conclusion, mAbs from both sets are reliable tools for the detection of SSTR1, 3 and 5, whereas the rabbit mAb against SSTR2A is recommended for use in routine clinical testing due to its superior binding affinity.

Citation Format: Chiara Lambertini, Patrizia Barzaghi-Rinaudo, Lisa D'Amato, Stefan Schulz, Paolo Nuciforo, Herbert A. Schmid. Comparative expression analysis of somatostatin receptor subtypes in human neuroendocrine tumors using two sets of new monoclonal antibodies. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 92. doi:10.1158/1538-7445.AM2013-92
Abstract 926: The angiotensin IV analog Norleual acts as a hepatocyte growth factor/c-Met inhibitor in pancreatic cancer cells.
Authors: Kawas Leen H. , Harris Phillip , Church Kevin , Wright John W. , Harding Joseph W.
Published: August 2014

The primary purpose of this study was to demonstrate that the hepatocyte growth factor (HGF) antagonist Norleual is capable of attenuating the cellular responses of pancreatic cancer cells to HGF. The angiotensin IV analog Norleual [Nle-Tyr-Ile-ψ-(CH2-NH2)3-4-His-Pro-Phe] exhibits structural homology with the hinge (linker) region of HGF and acts to block HGF dimerization, a process required for its activation. Norleual competitively inhibited the binding of a H3-Hinge peptide sequence to the HGF, and binds directly to HGF with a Ki = 3.6x10-12M. Predictably, Norleual is able to block the cellular responses due to HGF activation in multiple cell types at concentrations in the picomolar range including HGF/c-Met sensitive pancreatic cancer cells. Norleual's ability to inhibit the growth and survival of pancreatic cancer cells was evaluated using viability testing and cell sorting tags to identify living, dead and apoptotic cells. HGF/c-Met activation increases the invasive potential of the pancreatic cancer cells, which provides a mechanistic basis for the ability of c-Met to support pancreatic tumor metastasis. In this regard Norleual as an HGF antagonist was able to block the migration and invasion of pancreatic cancer cells through collagen gels in a transwell chamber. Over-activation of the growth factor system HGF/c-Met is a critical contributor to pancreatic cancer's ability to disseminate rapidly and its refractoriness to standard chemotherapy. Norleual, which is a representative of a “first-in-class” group of molecules that blocks the activation step of HGF, represent a novel therapeutic approach to the treatment of pancreatic cancer.

Citation Format: Leen H. Kawas, Phillip Harris, Kevin Church, John W. Wright, Joseph W. Harding. The angiotensin IV analog Norleual acts as a hepatocyte growth factor/c-Met inhibitor in pancreatic cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 926. doi:10.1158/1538-7445.AM2013-926
Abstract 937: Dissection of KRAS-driven survival signaling networks via phosphoproteomics in lung cancer cells.
Authors: Kim Jae-Young , Welsh Eric A. , Fang Bin , Oguz Umut , Li Jiannong , Kinose Fumi , Bronk Crystina , Beg Amer A. , Chen Ann , Eschrich Steven , Koomen John , Haura Eric B.
Published: August 2014

KRAS mutation accounts for development of approximately 30% of lung adenocarcinoma yet therapeutic options remain limited. We report our efforts to explore downstream signaling proteins driven by KRAS, including TANK-binding kinase 1 (TBK1), through a mass spectrometry based phosphoproteomics approach. We hypothesized that such a search would identify key survival proteins as well as uncover potential adaptive resistance mechanisms. We identified proteins whose phosphorylation is regulated TBK1 using SILAC and mass spectrometry following RNAi-mediated TBK1 knockdown. A cohesive network view of the underlying results centered major effects on Polo-like Kinase 1 (PLK1) and decreased phosphorylation of its targets implicating TBK1 as involved in mitosis. TBK1 activity was induced especially in late G1 and M phase, and loss of TBK1 sensitized lung cancer cells to a mitotic stressor. Surprisingly, we found that TBK1 knockdown increased phosphorylation of oncogenic kinases, including EGFR, Met, and ERK1/2. We screened multiple tyrosine kinase inhibitors in combination with TBK1 loss and found the pro-apoptotic effect of TBK1 loss was enhanced by dasatinib, a Src-family kinase inhibitor, providing a mechanistic basis for rational combinatorial therapies involving TBK1. Moving upstream, we analyzed global phosphoproteome change after KRAS knockdown and identified unexpected role of KRAS involved in CDK1 regulation as well as potential adaptive resistant mechanisms that can protect cells against KRAS loss. Finally, we have started exploring changes in the programmed kinome in KRAS mutated lung cancer cells following exposure to MEK inhibitors using activity based protein profiling. Initial experiments have characterized phenotypic effects of two MEK inhibitors in a subset of KRAS mutated lung cancer cells and preliminary studies have identified increased levels of some kinases following MEK inhibition. Collectively, we expect this kinome and phosphoproteomic network based approach will provide better insights into survival signaling mechanism maintaining survival of KRAS mutant lung cancer cell as well as novel therapeutic strategies for this cancer subtype. Updated work will be presented. Supported by the SPORE in Lung Cancer (P50-CA119997)

Citation Format: Jae-Young Kim, Eric A. Welsh, Bin Fang, Umut Oguz, Jiannong Li, Fumi Kinose, Crystina Bronk, Amer A. Beg, Ann Chen, Steven Eschrich, John Koomen, Eric B. Haura. Dissection of KRAS-driven survival signaling networks via phosphoproteomics in lung cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 937. doi:10.1158/1538-7445.AM2013-937
Abstract 942: Decreasing the AKT feedback loop with arsenic trioxide enhances mTORi anti-tumor effects.
Authors: Guilbert Cynthia , Annis Matthew G. , Siegel Peter , Miller Wilson H. , Mann Koren K.
Published: August 2014

Inhibitors of the mammalian target of rapamycin (mTORi) have exciting clinical activity in renal cell carcinoma, breast cancer, and some hematologic malignancies. However, the potential benefits of mTOR inhibition may be limited by a feedback mechanism that results in AKT activation. While mTORi can block important growth promoting events downstream from TORC1, an increased activation of AKT may inhibit apoptotic signals. Increased AKT activity resulting from mTOR inhibition is a result of increased growth factor/MAPK signaling or alternatively, via the second mTOR complex, TORC2. We have previously shown that arsenic trioxide (ATO) inhibits AKT activity and in some cases, decreases AKT protein expression. Therefore, we propose that combining ATO and mTORi may circumvent the AKT feedback loop and increase the anti-tumor effects of these drugs. Using a panel of breast cancer cell lines, we find that ATO, at clinically achievable doses, can enhance the inhibitory activity of the mTORi temsirolimus in some, but not all, cell lines. MCF-7, MDA-MB-468, and SKBR3, but not T47D cells, exhibit a decrease in cell number that correlates with an increase in the percentage of cells arrested in the G0/G1 phase of the cell cycle. In all these cell lines, temsirolimus treatment resulted in AKT activation, which was decreased by concomitant ATO treatment only in MCF-7, MDA-MB-468, and SKBR3 cell lines. Treatment with mTORi also results in activated ERK signaling, which is decreased with ATO co-treatment in MCF7, MDA-MB-468, and SKBR3. These data indicate that, when effective, ATO decreases phospho-AKT and ERK concomitantly. We next tested the toxicity and efficacy of rapamcyin plus ATO combination therapy in a MDA-MB-468 xenograft model. The combination was well-tolerated, and rapamycin did not increase ATO-induced liver enzyme levels. In contrast, the combination was significantly more effective at inhibiting tumor growth. The increased anti-tumor effects corresponded with ATO decreasing the rapamycin-induced phospho-Akt, phospho-ERK, and phospho-4EBP1 within the tumor. Therefore, we propose that adding ATO to mTORi treatment may overcome the negative feedback loop by decreasing MAPK and activated AKT in addition to the downstream targets of mTOR/TORC1.

Citation Format: Cynthia Guilbert, Matthew G. Annis, Peter Siegel, Wilson H. Miller, Koren K. Mann. Decreasing the AKT feedback loop with arsenic trioxide enhances mTORi anti-tumor effects. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 942. doi:10.1158/1538-7445.AM2013-942
Abstract 944: The effect of endogenous peroxidase activity on cell culture-based drug activity screening.
Authors: Bakhsheshian Joshua , Kwit Alexandra D.T. , Brimacombe Kyle R. , Hall Matthew D. , Gottesman Michael M.
Published: August 2014

Reactive oxygen species (ROS) are known to play an intrinsic role in mediating the cancer cell killing of many commonly employed anticancer agents. In many cases this is due to direct production of ROS via interaction with cellular components. This evidence is based partly on observations that ROS-scavenging agents such as N-acetylcysteine or catalase reverse killing in cancer cells. Catalase enzymatically degrades hydrogen peroxide (H2O2) into dioxygen and water. We examined the effect of catalase (5,000 U/mL) supplemented in growth media on the efficacy of ∼800 cytotoxic small molecules. Strong reversal of toxicity was found for 34 compounds with diverse mechanisms, including tubilin-interacting drugs. This implies that cytotoxins may have diminished efficacy in vivo due to the high activity of catalase in blood (∼100,000 U/mL). Surprisingly, we found that assays conducted with supplementation of catalase in media at levels greater than >20,000 U/mL, intended to replicate the endogenous environment, were highly toxic to cells. This phenomenon has been replicated in a number of cell lines from various tissue origins. One implication of this funding is that cancer cell lines require baseline ROS levels for cell survival proliferation, consistent with the cancer ROS addiction theory. Given that cell culture medium lacks peroxidase activity we conclude that the precise cell culture environment is a critical determinant of the results obtained when screening drugs for toxicity to cancer cells.

Citation Format: Joshua Bakhsheshian, Alexandra D.T. Kwit, Kyle R. Brimacombe, Matthew D. Hall, Michael M. Gottesman. The effect of endogenous peroxidase activity on cell culture-based drug activity screening. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 944. doi:10.1158/1538-7445.AM2013-944
Abstract 954: Multidrug resistance transcriptome analysis highlights compounds that sensitize resistant hepatocellular carcinoma through increased histone acetylation.
Authors: Gillet Jean-Pierre , Andersen Jesper B. , Madigan James P. , Varma Sudhir , Wu Chung-Pu , Calcagno Anna M. , Ambudkar Suresh V. , Thorgeirsson Snorri S. , Gottesman Michael M.
Published: August 2014

No effective systemic therapy exists for patients with advanced hepatocellular carcinoma (HCC). Surgery is the mainstay treatment for patients with early stage HCC tumors. Either resection or transplantation is advocated if the HCC is within the Milan criteria. Nonsurgical treatments including percutaneous ethanol injection, radiofrequency ablation, and transcatheter arterial chemoembolization (TACE) are used as adjuvant therapy to surgery but also to treat unresectable HCC. Although a meta-analysis showed a beneficial survival effect for patients with intermediate HCC treated by TACE using doxorubicin and cisplatin, the survival benefit of systemic chemotherapy for the treatment of liver cancer is marginal at best. Although Sorafenib, a multi-tyrosine kinase inhibitor, is becoming established as the first line of therapy for advanced HCC, it was recently shown to be a substrate of ABCB1 and ABCG2, two major transporters involved in multidrug resistance (MDR) and expressed in hepatocytes. In this study, the MDR transcriptome was analyzed in a cohort of 38 patients using TaqMan-based qRT-PCR, with the aim of finding ways to sensitize this intrinsically MDR cancer. The study reveals a 45-gene signature, validated using an independent cohort of 53 patients, that predicts overall survival (OS). We used the Connectivity Map tool, designed to reveal connections among drugs, genes, and pathological states, to identify three compounds that converted the gene expression profile of HCC cell lines from one matching patients with poor OS to one with good OS. These compounds increased histone acetylation, resulting in the sensitization of tumor cells to conventional chemotherapy including cisplatin, sorafenib and 5-FU. This work provides new treatment strategies based on gene expression profiling and connectivity data for a disease for which no effective systemic treatments exist. More importantly, the current study reveals a new strategy to sensitize any type of refractory cancer to chemotherapy.

Citation Format: Jean-Pierre Gillet, Jesper B. Andersen, James P. Madigan, Sudhir Varma, Chung-Pu Wu, Anna M. Calcagno, Suresh V. Ambudkar, Snorri S. Thorgeirsson, Michael M. Gottesman. Multidrug resistance transcriptome analysis highlights compounds that sensitize resistant hepatocellular carcinoma through increased histone acetylation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 954. doi:10.1158/1538-7445.AM2013-954
Abstract 962: Prolonged antiangiogenic therapy with oral metronomic topotecan and pazopanib in pediatric solid tumor mouse model.
Authors: Kumar Sushil , Mokhtari Reza Bayat , Islam Syed , Yeger Herman , Baruchel Sylvain
Published: August 2014

Background: We have previously observed that the combination of daily oral Low Dose Metronomic (LDM) Topotecan (TP) and Pazopanib (PZ) significantly delayed the tumor growth and enhanced the survival in neuroblastoma (NB) mouse models (Kumar et al, 2011). However, this combination (TP+PZ) did not stop tumor growth and the animals eventually died due to disease during the course of therapy. Therefore, we planned to investigate the factors contributing to resistance to this antiangiogenic therapy in NB mouse model.

Objectives: To investigate the effect of prolonged TP+PZ therapy on angiogenesis by testing the effect of therapy at different durations in NB mice xenograft model.

Methods: Mice bearing subcutaneous SK-N-BE(2) NB xenografts were treated with either of the four regimens, daily, orally: vehicle (untreated), PZ (150 mg pazopanib), TP (1.0 mg/Kg topotecan), TP+PZ (combination of topotecan and pazopanib, same doses as single agents). Durations of treatment planned for each regimen were 28 days, 56 days and 80 days. Animals were sacrificed after these durations or upon end point, whichever was earlier. The criteria for end point are tumor sizes exceeding 2.0 cms in diameter or animals showing signs of morbidity.

Results: The animals in control, PZ, TP reached the end point after 23d, 28d and 46d respectively. TP+PZ halted the tumor growth for up to 40 days, after which gradual growth was observed. The mean tumor diameters in the third TP+PZ group reached 1.8 cms at the day 80. CD31 immunofluorescence revealed that all the three durations of TP+PZ significantly lowered microvessel densities compared to the control (P= 0.02, 0.04 and 0.009 respectively for 28, 56 and 80 days respectively). However the ratio of CD31 to α- smooth muscle actin (pericyte marker) stained areas were significantly higher in animals treated with TP+PZ for 56 and 80days, compared to control (P=0.0006; 56d and 0.004; 80d) indicating higher pericyte coverage. TP+PZ (28d) showed signs of necrosis and cell cycle arrest (Ki67 staining), which were not observed in later durations (56 and 80 days) with TP+PZ. Long-term TP+PZ therapy also increased the levels of HIF-1 alpha, VEGF and Oct-4 within the tumor tissue.

Conclusion: This study indicates that even though prolonged TP+PZ therapy demonstrates sustained antiangiogenic activity, it encounters resistance in NB, potentially mediated by pericyte coverage. Long-term TP+PZ therapy may also increase tumor stemness.

Citation Format: Sushil Kumar, Reza Bayat Mokhtari, Syed Islam, Herman Yeger, Sylvain Baruchel. Prolonged antiangiogenic therapy with oral metronomic topotecan and pazopanib in pediatric solid tumor mouse model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 962. doi:10.1158/1538-7445.AM2013-962
Abstract 965: Establishment of a Sunitinib resistant cell line from ccRCC by long term drug application.
Authors: Unteregger Gerhard , Krein Simone , Weiland Anne , Stoeckle Michael , Junker Kerstin
Published: August 2014

Introduction: Sunitinib (SUN) acts as a multitargeted tyrosine kinase inhibitor (TKI) in the treatment of advanced renal cell carcinoma. It addresses predominantly the neo-angiogenic pathway by inhibition of proliferation and induction of apoptosis. But in clinical practice patients fail therapy. To elucidate the mechanism behind the failure of SUN we started to investigate the efficacy of SUN in long term cell culture studies using permanent cell lines representing ccRCC.

Material and Methods: CAKI-1/2, A-498 and 786-O cell lines, normal fibroblasts and HUVEC were subjected to different concentrations of SUN for up to 5 days. Efficacy of SUN was monitored by quantification of viability (WST-1), apoptosis (CDD+), necrosis (LDH+) and proliferation (BrdU). Under long term SUN-treatment growth behavior was assessed by cell counting spanning a cultivation period of several weeks. miRNA profiling was performed by microarray analysis at different point within this schedule.

Results: ccRCC cell lines offer a decrease in viability down to 40-60% at 2-5μM/L SUN dependent on cell-type and cell concentration as a result of f proliferation-inhibition. Endothelial cells and fibroblasts react more sensitive showing a growth inhibition between 80-90% compared to untreated controls. Long term application of SUN to these ccRCC-cells results in SUN-insensitive cell clones offering no inhibition of proliferation at 2-5μM/l drug. Specific miRNA expression changes were identified in insensitive cells.

Conclusion: Drug resistance by a long term SUN treatment provides cell lines to study the molecular mechanisms underlying this phenomenon. We have shown that resistance is associated with miRNA alterations. 3D-combination of these cell lines together with endothelial cells and fibroblasts offer a powerful tissue-like model to develop new therapeutic schedules to overcome the failure of actual treatment.

Citation Format: Gerhard Unteregger, Simone Krein, Anne Weiland, Michael Stoeckle, Kerstin Junker. Establishment of a Sunitinib resistant cell line from ccRCC by long term drug application. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 965. doi:10.1158/1538-7445.AM2013-965
Abstract 967: Establishing mechanisms of acquired resistance to Met inhibitor in Met-addicted gastric cancer cell lines.
Authors: Lai Andrea Z. , Thompson Crista , Cory Sean , Zhao Hong , Chughtai Naila , Hallett Michael , Park Morag
Published: August 2014

Signaling by the Met, hepatocyte growth factor (HGF) receptor tyrosine kinase (RTK) activates multiple downstream signaling pathways that promote cell migration and invasive growth. Cells that overexpress and are “addicted” to Met also require Met signaling to sustain cell survival. Thus, a number of specific small-molecule inhibitors have been developed to target Met in the clinic. Although several successes of RTK-targeted therapies are acknowledged, most have had limited long-term success clinically due to development of drug resistance.

As MET amplification occurs in 10-20% of gastric cancers, and MET-amplified cell lines are often exquisitely dependent on Met signaling for proliferation and survival, we established Met inhibitor-resistant clones derived from two different gastric cancer cell lines (MKN45 and KATO II) that are sensitive to Met inhibition. Treatment with a small-molecule inhibitor, PHA-665752 (PHA), specific to Met abrogates cell proliferation and anchorage-independent growth in these cells. In vitro selection of the most tumorigenic and resistant cells was performed by isolating colonies in anchorage-independent conditions with increasing PHA concentrations. Colonies were picked and expanded in 2D cell culture conditions and PHA concentration gradually increased until the cells were able to proliferate in a PHA concentration of >1μM (10x greater than the dose required for abrogation of proliferation in the parental cell lines).

DNA, RNA, and protein have been isolated from each of the clones and sequencing, microarray analyses, phospho-RTK arrays, reverse-phase protein arrays (RPPA) have been utilized to identify the changes in signalling, protein and transcript expression that allow these cells to escape PHA inhibition. The analyses from these high-throughput assays will be presented here.

As several Met inhibitors are presently in all three phases of clinical trials, and Met signalling has emerged as a means of escaping treatment by other targeted small-molecule kinase inhibitors, there exists a great need to determine the mechanisms through which cells may circumvent Met inhibition. The identification of resistance mechanisms will be essential for developing multi-pronged therapeutic strategies that may prove more efficacious at combatting tumorigenesis.

Citation Format: Andrea Z. Lai, Crista Thompson, Sean Cory, Hong Zhao, Naila Chughtai, Michael Hallett, Morag Park. Establishing mechanisms of acquired resistance to Met inhibitor in Met-addicted gastric cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 967. doi:10.1158/1538-7445.AM2013-967
Abstract 980: PD173074, a selective FGFR inhibitor, reverses ABCB1-mediated drug resistance in cancer cells.
Authors: Patel Atish S. , Tiwari Amit , Chufan Eduardo , Singh Satyakam , Sodani Kamlesh , Anreddy Nagaraju , Talele Tanaji , Ambudkar Suresh , Stephani Ralph , Chen Zhe-Sheng
Published: August 2014

Multidrug resistance (MDR) through overexpression of ATP-binding cassette (ABC) transporters has long evaded chemotherapy in cancer patients. Specific tyrosine kinase inhibitors (TKIs) were recently reported to modulate these ABC transporters, leading to an increase in the intracellular concentration of their substrate drugs. Here, we report for the first time, PD173074 an inhibitor of the fibroblast growth factor receptor (FGFR), to selectively inhibit ABC subfamily B member 1 (ABCB1/P-glycoprotein) transporter. PD173074 significantly sensitized both ABCB1 transfected and drug selected cell lines overexpressing this transporter to anticancer drugs colchicine, paclitaxel, and vincristine. This effect of PD173074 is specific, as no significant interaction was detected with other ABC transporters such as ABCC1, and ABCG2. The observed inhibition was primarily due to the decreased active efflux of [3H]-paclitaxel in ABCB1 overexpressing cells. However, no significant change in the ABCB1 expression was observed when ABCB1 overexpressing cells were exposed to 5 μM PD173074 for up to 3 days thereby suggesting its role in modulating the function of the transporter. In addition, PD173074 stimulated the ATPase activity of ABCB1 in a concentration-dependent manner, indicating a direct interaction with the transporter. Interestingly, PD173074 did not inhibit the photolabeling of ABCB1 with [125I]-Iodoarylazidoprazosin (IAAP) showing that it binds at a site different from that of IAAP in the drug-binding pocket. In conclusion, PD173074 could selectively reverse ABCB1-mediated MDR by directly blocking the efflux function of ABCB1.

Citation Format: Atish S. Patel, Amit Tiwari, Eduardo Chufan, Satyakam Singh, Kamlesh Sodani, Nagaraju Anreddy, Tanaji Talele, Suresh Ambudkar, Ralph Stephani, Zhe-Sheng Chen. PD173074, a selective FGFR inhibitor, reverses ABCB1-mediated drug resistance in cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 980. doi:10.1158/1538-7445.AM2013-980
Abstract 990: Efficacy of various chemotherapeutic agents on 5-FU-resistant human gastric cancer cell lines and their mechanisms.
Authors: Nukatsuka Mamoru , Kobunai Takashi , Nakamura Ayako , Hayashi Kazuhiko , Takechi Teiji
Published: August 2014

Purpose: Four 5-fluorouracil (5-FU)-resistant human gastric cancer cell lines were established by the long-term exposure of parent cell lines (MKN45, MKN-74, KATO-III, and NCI-N87) to 5-FU. The cytotoxicity and mechanisms of several chemotherapeutic agents were then investigated using 5-FU-resistant gastric cancer cell lines.

Method: 5-FU-resistant cell lines were established by continuous exposure to escalating concentrations (1-5 μM) of 5-FU over a 1-year period. The sensitivities of the cell lines to chemotherapeutic agents (paclitaxel, docetaxel, cisplatin, oxaliplatin, epirubicin, and SN-38) were then evaluated using the WST-8 colorimetric assay. To elucidate the mechanisms of resistance, the gene expressions and copy numbers were measured using an Agilent Technologies whole human genome oligo DNA microarray and a CGH microarray. The protein and mRNA expressions of a target enzyme of 5-FU, thymidylate synthase (TS), were also investigated using western blotting and RT-PCR, respectively.

Results: The IC50 ratios of the 5-FU-resistant cell lines were 2.6 to 15.6 times higher than those of the parent cell lines in vitro. MKN-45/5-FU showed cross-resistance to cisplatin and oxaliplatin and KATO-III/5-FU showed cross-resistance to SN-38, while MKN-74/5-FU showed collateral sensitivities to docetaxel, cisplatin, and oxaliplatin, and NCI-N87/5-FU showed collateral sensitivities to cisplatin, oxaliplatin, SN-38, and epirubicin. RT-PCR showed that TS mRNA expression was higher in the parent cell lines than in the 5-FU-resistant cell lines, and the TS protein expression level was also higher in all the resistant cell lines. For MKN-45/5-FU and KATO-III/5-FU, not only the mRNA expression level but also the TS gene copy number was increased. Interestingly, the expression of MAP3K15, which is an apoptosis-facilitating factor, increased in NCI-N87/5-FU, which showed collateral sensitivity to cisplatin, oxaliplatin, SN-38, and epirubicin.

Conclusion: Analyzing the molecular mechanisms of 5-FU-resistant cell lines may be useful for the development of effective chemotherapy regimens for relapsed gastric cancer after 5-FU-based chemotherapy.

Citation Format: Mamoru Nukatsuka, Takashi Kobunai, Ayako Nakamura, Kazuhiko Hayashi, Teiji Takechi. Efficacy of various chemotherapeutic agents on 5-FU-resistant human gastric cancer cell lines and their mechanisms. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 990. doi:10.1158/1538-7445.AM2013-990
Abstract 992: Regulation of ABCG2 lysosomal degradation by xanthines: Role of the PI3K/AKT pathway.
Authors: Ding Rui , Kamen Barton A. , Shi Jia , Scotto Kathleen W.
Published: August 2014

ABCG2 confers multidrug resistance to a wide range of structurally unrelated chemotherapeutic drugs, often resulting in the failure of cancer chemotherapy. Accordingly, the downregulation of ABCG2 expression and/or function has been proposed as part of a regimen to improve cancer therapeutic efficacy. Previously, we have reported that a group of xanthines including caffeine, theophylline and dyphylline can dramatically decrease ABCG2 protein in drug resistant cells, causing a significant increase in the intracellular retention of ABCG2 substrates and sensitization of the cells to chemotherapeutic agents (Ding et al., Mol Pharmacol 81:328-337, 2012). This downregulation of ABCG2 is due to a selective acceleration of its lysosomal degradation by xanthines. We now show that xanthines induce the endocytosis and subsequent degradation of ABCG2. In addition, PI3K/AKT/mTORC1 signaling was found to be involved in mediating the effect of xanthines on ABCG2, since both caffeine and theophylline strongly inhibited activation of AKT and mTORC1, and inhibition of both AKT and mTORC1 mimicked the effect of xanthines on ABCG2. Furthermore, adenosine, a nucleoside vital for purine metabolism and energy homeostasis, fully reversed the downregulation effect of caffeine on ABCG2. Interestingly, this adenosine-mediated effect occurs intracellularly and independent of adenosine receptor signalling, identifying a novel mechanism whereby the xanthine-degradation of ABCG2 is influenced by intracellular levels of adenosine. Importantly, adenosine also blocked the lysosomal degradation of ABCG2 that was induced by PI3K/AKT inhibition, indicating that the adenosine-mediated intracellular events cross-talk with the PI3K/AKT pathway in regulating ABCG2 degradation. This study enhances our understanding of the regulation of ABCG2 internalization and degradation, and identifies new targets for the development of ABCG2 expression/function modulating agents for chemosensitization.

Citation Format: Rui Ding, Barton A. Kamen, Jia Shi, Kathleen W. Scotto. Regulation of ABCG2 lysosomal degradation by xanthines: Role of the PI3K/AKT pathway. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 992. doi:10.1158/1538-7445.AM2013-992
Abstract ADV08: Bridging the gaps in service.
Authors: Dauria Mary Kay
Published: August 2014

Susan's Rally is a Houston based 501c3 organization that started as a group supporting breast cancer research and has expanded to support services provided for those with all types of cancer. What began as a fun road rally for Bruce and Susan Roberson morphed into a fun-raising event for cancer after Susan was taken by breast cancer. For 17 years, Bruce and his friends have continued the rally in her honor and now the proceeds from the rally, golf tournament, and auctions are used to fight the disease and assist with providing patients support services.

Susan's Rally has raised over a million dollars in cash and in-kind donations utilizing all volunteer labor. Except for a few office supplies, postage and printing, all of Susan's Rally dollars go to fight cancer and support patients. Some of the programs supported included MD Anderson Patients Support Network, research at the Christus Stehlin Foundation for Cancer Research, provide diagnostic imaging for uninsured women at The Rose, counseling at Y-Me, and assists with making available low-cost short-term housing at The Sojourn House-Apartment, homes for cancer patients. In 2012 Susan's Rally also began funding “Dia De la Mujer community health festivals providing free breast exams, HIV testing an pap smears for women in need.

Citation Format: Mary Kay Dauria. Bridging the gaps in service. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr ADV08. doi:10.1158/1538-7445.AM2013-ADV08
Abstract ADV09: Bladder cancer basics.
Authors: Hoffman Vanessa
Published: August 2014

The Bladder Cancer Advocacy Network's (BCAN) mission is to increase public awareness about bladder cancer, to advance bladder cancer research, and to provide educational and support services for the bladder cancer community. BCAN works to serve over 520,000 bladder cancer survivors and 73,000 newly diagnosed patients each year in the United States. BCAN engages in many initiatives and programs. BCAN's current initiatives include the Women's Outreach Initiative and Veterans’ Outreach Initiative, which are engaging national task forces to raise awareness of bladder cancer among these constituencies.

In patient education and support, BCAN's website: www/bacan.org- is one of the most comprehensive bladder cancer resources on the internet. BCAN also provides free bladder cancer patient education materials including patient tip sheets and a patient handbook-Bladder Cancer Basics for the Newly Diagnosed, which was revised this year and is now available in Spanish. BCAN offers Patient Insight Webinars throughout the year. BCAN also provides a free clinical trial matching service in coordination with Emerging Med, to help people obtain specific information about participating in bladder cancer research trials. BCAN staff answers patient calls and e-mails for more information, resources, and support. BCAN has facilitated the development of in-person bladder cancer support groups around the country and offers access to an online bladder cancer support community.

BCAN also offers the Young Investigator Awards, announced this year, for three awards of $100,000 over two years, to increase the understanding of bladder cancer risk, biology, prevention, and treatment while encouraging young investigators to pursue a career in bladder cancer research. BCAN is also engaged in advocacy with staff members and volunteers serving on panels and participating in coalitions. BCAN has coordinated Walks for Bladder Cancer to raise awareness of the disease since 2011, on the first Saturday in May. In 2012, the Walks for Bladder Cancer held on May 5 took place in 60 communities, had more than 5,000 participants and raised more than $275,000.

Citation Format: Vanessa Hoffman. Bladder cancer basics. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr ADV09. doi:10.1158/1538-7445.AM2013-ADV09
Abstract LB-100: Exploring the mechanisms of resistance to Salinomycin, a cancer stem cell-specific agent, in budding yeast as a model.
Authors: Rozario Donald , Zeglam Sammer , Siede Wolfram
Published: August 2014

Introduction

The antibiotic salinomycin (SM) represents a selective potassium ionophore. It has recently attracted considerable attention due to its stem-cell specific mode of action that has been elucidated in a breast cancer stem cell model [1]. Further studies revealed additional benefits, e.g. the triggering of apoptosis in resistant cancer cell lines and reversal of multidrug resistance [2,3]. In budding yeast, we discovered that this agent prominently causes mitochondrial damage as revealed by the emergence of respiration-defective cells (“petites”), similar to nigericin. We also noted the frequent occurrence of SM-resistant clones - a possible obstacle in therapy.

Methods

We used the collection of systematic gene deletions in haploid Saccharomyces cerevisiae to characterize genes whose deletion confers SM sensitivity or resistance if a carbon source is provided (glycerol) that requires active respiration for growth. In parallel, the emergence of reactive oxygen species (ROS) was measured by flow cytometry. With this toxicogenomics approach in a model organism, we attempt to elucidate cancer-stem cell specific therapy targets and suggest strategies to avoid the development of salinomycin resistance.

Results and Conclusions

Among SM-sensitive deletion mutants, mutants that compromise vacuole/Golgi function and autophagy dominate. Novel genes which confer SM sensitivity if deleted were also uncovered, such as CAJ1, a heat shock protein of the dnaJ family. In almost all cases, a strong correlation between SM and nigericin sensitivity was observed.

Among the most SM-resistant mutants were deletions of the E2-ubiquitin conjugating enzymes Ubc13 and Mms2 as well as deletions of TOR pathway components Sch9 and Tco89. In the presence of SM, Sch9 (homolog of human Akt) underwent dephosphorylation; a deletion of Sch9 was shown to improve growth in glycerol media and to counteract petite induction in glucose media in SM-treated cells. The observed mitochondria-dependent ROS production by SM, however, was unaltered and we suggest that, while initial damage is not affected, cells acquire resistance to the consequences of such damage.

[1] Gupta PB, Onder TT, Jiang G, Tao K, Kuperwasser C, et al. (2009) Cell 138: 645-659. [2] Fuchs D, Heinold A, Opelz G, Daniel V, Naujokat C (2009) Biochem Biophys Res Commun 390: 743-749. [3] Fuchs D, Daniel V, Sadeghi M, Opelz G, Naujokat C (2010) Biochem Biophys Res Commun 394: 1098-1104.

Supported by Cancer Prevention and Research Institute of Texas.

Citation Format: Donald Rozario, Sammer Zeglam, Wolfram Siede. Exploring the mechanisms of resistance to Salinomycin, a cancer stem cell-specific agent, in budding yeast as a model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-100. doi:10.1158/1538-7445.AM2013-LB-100
Abstract LB-108: Exosome profiling of mRNAs and miRNAs in serum samples from GBM patients .
Authors: Skog Johan , Bentink Stefan , LoGuidice Lori , Romain Charlotte , Belzer Susan , Noerholm Mikkel
Published: August 2014

Background: Biomarker discovery and development in oncology has traditionally required the use of tissue, either FFPE or frozen. In recent years, the use of Circulating Tumor Cells (CTCs), cell free DNA (cfDNA), and most recently, exosomes, has allowed biomarker research in biofluids to evolve. Exosomes contain mRNA, microRNA, DNA and protein in a protected, highly stable vesicle. Recently, we have shown that large panels of mRNA genes can be run on small samples of frozen, archived serum. The ability to assess the expressions patterns in blood represents a potentially important development for biomarker research and the development of non-invasive diagnostics.

Methods: Frozen, archived serum (2ml) was obtained from a glioblastoma clinical trial cohort, containing serum from both responders and non-responders to a novel therapy. We isolated the microvesicle fraction from the serum, extracted the RNA, and qualified the RNA using an Agilent 2100 Bioanalyzer on an Agilent 6000 RNA Picochip. A standard in-house control extraction was processed in parallel and subjected to quality control on the Bioanalyzer as well as RT-qPCR. The first cohort of samples (n = 60) were reverse transcribed to cDNA, and the cDNA was assayed on a human cancer mRNA panel Array by qPCR (Life Technologies).The cancer panel includes a total of 648 cancer related genes including endogenous controls. The second cohort of samples (n = 40) was reverse transcribed and analyzed by a miRNA panel of 758 different miRNA assays and controls.

Results: Our data clearly demonstrate the ability to detect relevant gene expression patterns in microvesicle RNA isolated from patient serum. High quality RNA was extracted from the samples received, and an average of 80% of the 648 cancer associated mRNA genes and 36% of the miRNAs were detected in the samples with the volumes used in this study. Data was demonstrated to be reproducible between and among samples and suggests a biological pattern of RNAs in serum exosomes that could be used to monitor response to treatment.

Conclusions: RNA isolated from the microvesicle fraction of serum contains abundant, high-quality RNA, including mRNA. The analysis of a large panel of genes from a small sample of serum is possible, demonstrating that RNA isolated from microvesicles from peripheral blood has the potential to be used as a source of genetic material for non-invasive diagnostic tests.

Citation Format: Johan Skog, Stefan Bentink, Lori LoGuidice, Charlotte Romain, Susan Belzer, Mikkel Noerholm. Exosome profiling of mRNAs and miRNAs in serum samples from GBM patients . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-108. doi:10.1158/1538-7445.AM2013-LB-108
Abstract LB-115: Cooperative activation of tissue specific genes by pRB and E2F1.
Authors: Flowers Stephen J. , Xu Fuhua , Moran Elizabeth
Published: August 2014

The retinoblastoma tumor suppressor protein pRB is conventionally regarded as an inhibitor of the E2F family of transcription factors. Conversely, pRB is also recognized as an activator of tissue-specific gene expression along various lineages including osteoblastogenesis. During osteoblast differentiation, pRB directly targets Alpl and Bglap, which encode the major markers of osteogenesis, alkaline phosphatase and osteocalcin. Surprisingly, p130 and repressor E2Fs were recently found to co-occupy and repress Alpl and Bglap in proliferating osteoblast precursors prior to differentiation. This raises the further question of whether these genes convert to E2F activation targets when differentiation begins, which would constitute a remarkable situation wherein pRB and E2F would be co-targeting genes for activation.Chromatin immunoprecipitation (ChIP) analysis in an osteoblast differentiation model shows Alpl and Bglap are indeed targeted by an activator E2F, i.e. E2F1. Promoter occupation of Alpl and Bglap by E2F1 occurs specifically during activation, and depletion of E2F1 severely impairs their induction. Mechanistically, promoter occupation by E2F1 and pRB is mutually dependent, and without this cooperative effect, activation steps previously shown to be dependent on pRB, including recruitment of RNA polymerase II, are impaired. Myocyte and adipocyte specific genes are also co-targeted by E2F1 and pRB during differentiation along their respective lineages. The finding that pRB and E2F1 cooperate to activate expression of tissue-specific genes is a paradigm distinct from the classical concept of pRB as an inhibitor of E2F1, but is consistent with the observed roles of these proteins in physiological models.

Citation Format: Stephen J. Flowers, Fuhua Xu, Elizabeth Moran. Cooperative activation of tissue specific genes by pRB and E2F1. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-115. doi:10.1158/1538-7445.AM2013-LB-115