bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5049: Targeting the Notch and mTOR pathways in diffuse intrinsic pontine glioma.
Authors: Hutt Marianne , Goldstein Wendy , Nazarian Javad , Price Antoinette , Lim Kah Jing , Warren Katherine , Chang Howard , Eberhart Charles , Raabe Eric
Published: August 2014

Diffuse intrinsic pontine glioma (DIPG) is a universally fatal pediatric brain tumor which is known to have frequent alteration of mTOR signaling. Analysis of DIPG tissue obtained through the Mid-Atlantic DIPG Consortium (MADC) also shows high level activation of Notch pathway effectors HES4 and HES5. We have established JHH DIPG1, a human DIPG xenograft and neurosphere cell line from a rapid autopsy specimen obtained through the MADC. Notch 1 and 3 and Notch downstream effectors, Hes1, Hes5 and Hey1, are highly expressed in JHH DIPG1 and a second DIPG line, SU DIPG1. These DIPG cell lines also express high level phospho-S6 and phospho-AKT 473, indicating activation of TORC1 and TORC2. Notch and mTOR pathway signaling is equal to or exceeding that observed in glioblastoma neurosphere cell lines. Notch targeting using a gamma-secretase inhibitor leads to a dose-dependent reduction in cell proliferation of up to 30% (p=0.05) as well as inhibition of phospho-AKT 473, suggesting that inhibition of the NOTCH pathway targets TORC2 complexes. BrdU incorporation also showed that treatment of DIPG cell lines with a TORC1 inhibitor also has up to a 60% attenuation of cell proliferation (p<0.005) in treated DIPG cells compared to non-treated cells and a dose-dependent reduction in phospho-S6, with concomitant upregulation of phospho-AKT 473 indicating TORC2 activation. Dual TORC1 and TORC2 inhibition by combining a rapalog and a gamma-secretase inhibitor may prove to be synergistic in DIPG. Simultaneous treatment with a rapalog and gamma-secretase inhibition suppresses TORC1 and TORC2, as measured by western blot. We are currently investigating the possible mechanism of action of these drugs in DIPG. Our laboratory is investigating additional metabolic inhibitors that can be used with TORC1 and TORC2 inhibitors to further improve treatment options for DIPG. We are currently exploring the role of the Notch and mTOR pathways in DIPG in pre-clinical studies in mice, with the goal of moving targeted inhibitors into clinical trials for these currently highly lethal tumors.

Citation Format: Marianne Hutt, Wendy Goldstein, Javad Nazarian, Antoinette Price, Kah Jing Lim, Katherine Warren, Howard Chang, Charles Eberhart, Eric Raabe. Targeting the Notch and mTOR pathways in diffuse intrinsic pontine glioma. [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 5049. doi:10.1158/1538-7445.AM2013-5049
Abstract 5053: Sonic hedgehog inhibition reduces in vitro tumorigenesis and alters expression of GLI1-target genes in a desmoplastic medulloblastoma cell line.
Authors: Garcia-Lopez R , Vera-Cano B , Vacas-Oleas A , de la Rosa J , Gallo-Oller G , Shahi M H. , Fan X , Alonso M M. , Rey J A. , Castresana Javier S.
Published: August 2014

Medulloblastoma is one of the most frequent and aggressive tumors of childhood. The Sonic hedgehog (Shh) pathway, related to human development, is altered in most medulloblastomas: genes like Ptch, Smo, or Sufu suffer mutations in 15% to 25% of these tumors. We tested Shh inhibition in the Daoy medulloblastoma cell line by two methods: a molecular one (direct Gli1 siRNA inhibition); and a pharmacological inhibition of Smo, upstream of Gli1, by cyclopamine. Afterwards, a comparison of cellular and molecular responses was done. We proved that MTT cell viability, and cell migration assessed by the scratching assay decreased after Shh inhibition. Furthermore, colony formation assay in culture decreased by 70%, and colony formation assay in soft agar decreased up to 90% when Shh inhibition was applied. As a whole, Shh inhibition conferred a less in vitro tumorigenic status to Daoy cells. Moreover, we assessed the expression of different Gli1 target genes and other genes, before and after Shh inhibition, and found that Shh shows a crosstalk with oncogenes and tumor suppressor genes that have been described in numerous tumors. Therefore, we found downregulation of Ptch1, Cyclin D2, Plakoglobin, Nkx2.2, Bmi1, Smo and N-myc after Shh inhibition. Sufu and Gli3 showed parallel results, where Gli1 siRNA did neither decrease nor increase expression of both genes, whereas cyclopamine reduced them in 15-25%. Pax6 mRNA levels were upregulated by either Gli1 siRNA or cyclopamine. Finally, Notch1 was upregulated after inhibition of Shh, while Notch2 showed contrasting results, as siRNA inhibition decreased its expression, while cyclopamine increased it. All these experiments give an overview of the Shh pathway in medulloblastoma, its relationship with other genes, and the demonstration of the efficacy of cyclopamine and Gli 1 siRNA Shh inhibition in vitro.

Citation Format: R Garcia-Lopez, B Vera-Cano, A Vacas-Oleas, J de la Rosa, G Gallo-Oller, M H. Shahi, X Fan, M M. Alonso, J A. Rey, Javier S. Castresana. Sonic hedgehog inhibition reduces in vitro tumorigenesis and alters expression of GLI1-target genes in a desmoplastic medulloblastoma cell line. [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 5053. doi:10.1158/1538-7445.AM2013-5053
Abstract 5070: Cell-free endo180 reveals additional level of endocytic receptor functioning in meningioma.
Authors: Gogineni Venkateswara Rao , Nalla Arun Kumar , Velpula Kiran Kumar , Rao Jasti S.
Published: August 2014

Endo180, also known as uPAR associated protein (uPARAP), is a member of the tri-molecular complex formed by uPA along with uPAR in the endocytic processing of collagen degradation. In the present study, we found that this membrane-bound protein was released into culture supernates of meningioma and other cancer cell types. In addition, we report a tremendous increase of Endo180 transcript and protein levels in irradiated meningioma cells and their culture filtrates. While non-denaturing immunoprecipitation studies suggested that Endo180 interacts with uPA in cell-free conditions, uPA binding was significantly quenched by deglycosylation of solubilized Endo180. Myc-tagged cDNA expression suggested shedding of Endo180 against secretion from meningioma cells. Function blocking and knockdown of Endo180 adversely affected the proliferation of meningioma cells with concomitant decrease in uPA activity and uPAR expression. We also observed a significant rise of Endo180 expression in meningioma clinical samples and tumors in animal models. A further increase of expression was observed among the brain sections of mice infused with irradiated meningioma cells. Taken together, our results demonstrate that solubilization of Endo180 characterizes a different hierarchy of endocytic receptor functioning in meningioma.

Citation Format: Venkateswara Rao Gogineni, Arun Kumar Nalla, Kiran Kumar Velpula, Jasti S. Rao. Cell-free endo180 reveals additional level of endocytic receptor functioning in meningioma. [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 5070. doi:10.1158/1538-7445.AM2013-5070
Abstract 5076: Identification and anti-angiogenic effects of small molecule inhibitors of TEM8.
Authors: Cryan Lorna , Roznik Marinya , McBride Aaron , D'Amato Robert , Christensen Kenneth , Rogers Michael S.
Published: August 2014

Tumor endothelial marker 8 (TEM8) is a gene upregulated on colonic tumor endothelium compared to quiescent normal endothelium. Inhibition of TEM8 using antibodies potently inhibits the growth of a variety of tumor types in mice. This inhibition of tumor growth is accompanied by a reduction in microvessel density within the tumors. Thus, TEM8 appears to represent a tumor specific endothelial marker that has a functional role in tumor angiogenesis. TEM8 is also a receptor for anthrax toxin. In the current study, we used chemical libraries to identify small molecule inhibitors of TEM8 that may have efficacy as inhibitors of tumor angiogenesis and anthrax toxin.

We conducted a fluorescence resonance energy transfer (FRET) based screen of over 270,000 small molecules in order to identify molecules that inhibit the interaction of TEM8 with its well-known ligand anthrax protective antigen (PA). We describe here the identification and anti-angiogenic effects of small molecules that bind to TEM8.

From the initial screen 280 molecules inhibited TEM8/PA interaction by greater than 50%. A secondary screen involved the elucidation of IC50 values for these 280 compounds as inhibitors of the TEM8/PA interaction. Twelve compounds had measurable IC50s in the micromolar range. The anti-angiogenic effects of these compounds were studied in vitro. Of the 12 compounds tested in a human microvascular endothelial cell (HMVEC) proliferation assay, 3 compounds significantly inhibited endothelial cell proliferation after 24 hours (P <0.05), and 5 compounds significantly inhibited proliferation at 72 hours (P <0.05). None of the compounds exhibited measurable cytotoxicity. One of the five compounds tested for effects on HMVEC migration, significantly inhibited migration on a fibronectin coated-surface by 78% (P<0.01).

We have identified a number of small molecule inhibitors of TEM8, some of which display anti-angiogenic effects in vitro. Should these inhibitors also display potent anti-angiogenic effects in vivo, they may serve as lead compounds for the future development of TEM8 therapeutic small molecules. Future studies will focus on determining the effects of these molecules on tumor growth and angiogenesis in mouse models.

Citation Format: Lorna Cryan, Marinya Roznik, Aaron McBride, Robert D'Amato, Kenneth Christensen, Michael S. Rogers. Identification and anti-angiogenic effects of small molecule inhibitors of TEM8. [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 5076. doi:10.1158/1538-7445.AM2013-5076
Abstract 5092: Ziv-aflibercept treatment reduces intra-hepatic tumor growth of a preclinical colorectal carcinoma liver metastasis model, monitored using non-invasive micro-ultrasound and bioluminescent imaging.
Authors: Adler Alexander P. , Parveen Asma , Thurston Gavin , Eichten Alexandra
Published: August 2014

The liver is a frequent metastatic site for different types of cancers, including colorectal carcinomas (CRC). Therapies that inhibit the angiogenic growth factor VEGF, such as bevacizumab and ziv-aflibercept, are used in combination with chemotherapies to treat metastatic CRC. Ziv-aflibercept (also known as VEGF Trap) is being developed as a part of a collaboration between Sanofi and Regeneron Pharmaceuticals, Inc. and was recently approved by the FDA in metastatic colorectal cancer that is resistant to or has progressed following an oxaliplatin-containing regimen. To better understand the role of VEGF in metastatic CRC, we aimed to assess the effects of ziv-aflibercept on intrahepatic CRC tumor growth, invasion and angiogenesis in a preclinical model. For that we developed and characterized a CRC liver metastasis model that would allow us to longitudinally assess the effects of ziv-aflibercept on CRC growth in the liver parenchyma using non-invasive high frequency micro-ultrasound and bioluminescent imaging modalities. Briefly, luciferase-expressing HCT116 (human colorectal carcinoma) tumor cells were implanted into the left liver lobe of male CB17 SCID mice using image-guided micro-ultrasound. Tumor growth was monitored using both micro-ultrasound and bioluminescent imaging. When tumors were established (approximately 70 mm3), mice were treated with either control protein (hFc, 25 mg/kg, 2x/wk) or ziv-aflibercept (25 mg/kg, 2x/wk) for up to 13 days. Ziv-aflibercept treatment inhibited tumor growth by 95% compared to control treatment. This inhibition of tumor growth was accompanied by significantly decreased cell proliferation (50-70%), as measured by Ki67 immunohistochemistry, and markedly decreased vessel density (approximately 50%), as revealed by CD31 immunohistochemistry. Ziv-aflibercept did not seem to increase tumor invasiveness, as H&E histology did not reveal dramatic differences at the tumor/stroma border between the ziv-aflibercept and control treated tumors. These experiments confirm that ziv-aflibercept reduces growth and vascularity of colorectal tumors growing in the liver in a preclinical model.

Citation Format: Alexander P. Adler, Asma Parveen, Gavin Thurston, Alexandra Eichten. Ziv-aflibercept treatment reduces intra-hepatic tumor growth of a preclinical colorectal carcinoma liver metastasis model, monitored using non-invasive micro-ultrasound and bioluminescent imaging. [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 5092. doi:10.1158/1538-7445.AM2013-5092
Abstract 5094: Angiopoietin-2 antagonist enhances the anti-angiogenic effect of sunitinib in a preclinical renal cell carcinoma model.
Authors: Molnar Nikolett , Siemann Dietmar W.
Published: August 2014

Angiogenesis, the formation of blood vessels from pre-existing ones, is an essential process in tumor growth and development. During angiogenesis, normal vessels consisting of endothelial and peri-endothelial cells are first destabilized via the Angiopoietin-2 (Ang-2)-Tie2 axis, and then endothelial cells are activated to proliferate and form new vessels mainly through the Vascular Endothelial Growth Factor (VEGF)-VEGFR2 axis. Currently there are several anti-angiogenic therapies targeting the VEGF-VEGFR pathway used in the clinic in various cancer settings. New therapies such as Ang-2 antagonists are also being pursued and have recently been shown to be valuable especially in combination with anti-VEGF therapies. Renal cell carcinoma (RCC) is a highly vascularized disease where anti-angiogenic therapies are used as both first and second line treatments. The present study focused on evaluating the effects of an Ang-2 antagonist in combination with the FDA approved Sunitinib, a first line treatment in RCC. An in vivo intradermal assay with VHL mutant, human renal cell carcinoma, Caki-2, cells was used to assess angiogenesis of either the Ang-2 antagonist, Sunitinib or the combination of the Ang-2 antagonist and Sunitinib. Results show a greater decrease in the number of tumor cell induced vessels in the Ang-2 and Sunitinib combination group compared to either Ang-2 antagonist or Sunitinib alone. Tumor growth was also impaired by these agents when used alone or in combination; however there was greater decrease in tumor volume in the Ang-2 and Sunitinib combination group compared to either Ang-2 antagonist or Sunitinib alone. These data show greater tumor vessel and tumor inhibition when an Ang-2 antagonist and Sunitinib are administered in combination.

Citation Format: Nikolett Molnar, Dietmar W. Siemann. Angiopoietin-2 antagonist enhances the anti-angiogenic effect of sunitinib in a preclinical renal cell carcinoma 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 5094. doi:10.1158/1538-7445.AM2013-5094
Abstract 5111: Protective effect of Traditional Chinese Medicine formula LQ against cyclophosphamide-induced renal damage in nude mice.
Authors: Zhang Lei , Wu Chengyu , Hoffman Robert M.
Published: August 2014

TCM formula LQ is an herbal cocktail. Cyclophosphamide (CYC) is widely used in cancer chemotherapy. However renal damage is a dose-limiting side effects of CYC. In the current study, we determined if LQ can protect against damage. The heart, liver, spleen, lung, kidney, and intestine of a mouse from control, LQ pretreatment, CYC pretreatment, and CYC+LQ treatment were stained with HE. The histology of each organ was observed under microscopy. The histology of the heart, liver, spleen, lung and intestine showed normal histopathological structure in all groups. The kidneys in the control mice, LQ pretreated mice, and CYC+LQ pretreated mice showed normal architecture of kidney cortex and medulla. The cortex showed normal renal corpuscle, proximal convoluted tubules, and distal convoluted tubules. The inner medulla and outer medulla all contained a thick ascending limb of the loop of Henle, and interstitial connective tissue. However, kidney of the CYC pretreatment mice showed damage in structures compare with other groups. The toxic effect of CYC on the kidney involved the glomeruli, the tubules, and the interstitium. The structures of the cortex in CYC pretreated group showed disconnection between glomeruli and tubules. The ducts in the medulla were destroyed. LQ also protected against weight loss and cachexia induced by CYC. LQ may be a promising drug for combination with CYC to improve its therapeutic index.

Citation Format: Lei Zhang, Chengyu Wu, Robert M. Hoffman. Protective effect of Traditional Chinese Medicine formula LQ against cyclophosphamide-induced renal damage in nude mice. [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 5111. doi:10.1158/1538-7445.AM2013-5111
Abstract 5112: Traditional Chinese medicine formula LQ inhibits spontaneous and cyclophosphamide-enhanced metastasis.
Authors: Zhang Lei , Wu Chengyu , Zhang Yong , Hoffman Robert M.
Published: August 2014

TCM (Traditional Chinese Medicine) formula LQ, an herbal cocktail, has shown anti-metastasis and anti-tumor efficacy without overt side-effects. In the present study, the human HT1080 fibrosarcoma cell line was labeled with both red fluorescent protein (RFP) in the cytoplasm and green fluorescent protein (GFP) in the nucleus. Dual-color HT1080 cells were injected into the footpad of mice following LQ treatment. Dual-color HT1080 cells were also injected into epigastric cranialis vein of the mice pre-treated with cyclophosphamide (CYC) or a combination of CYC and LQ. The efficacy was monitored through dynamic subcellular imaging of trafficking of cancer cells in lymph nodes and blood vessels in live mice. LQ significantly inhibited HT1080 spontaneous metastases to their local lymph nodes in the foot pad injection model. In the control group, 100% of mice had lymph node metastasis, compared to 12.5% of the mice in the LQ group. LQ also inhibited cyclophosphamide-enhanced experimental metastasis in the epigastric cranialis vein injection model. LQ prolonged the overall survival of mice in the spontaneous lymph node metastatic model. LQ therefore shows promise as an antimetastatic agent.

Citation Format: Lei Zhang, Chengyu Wu, Yong Zhang, Robert M. Hoffman. Traditional Chinese medicine formula LQ inhibits spontaneous and cyclophosphamide-enhanced metastasis. [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 5112. doi:10.1158/1538-7445.AM2013-5112
Abstract 5121: Differential expression of CC chemokine receptor-9 in prostate cancer.
Authors: Singh Rajesh , Gupta Pranav , Grizzle William E. , Lillard James W. , Singh Shailesh
Published: August 2014

Despite recent advances in diagnosis and treatment, prostate cancer is the second leading cause of cancer-related deaths in US. Precise etiology and molecular target to treat prostate cancer is yet to be defined. In recent years, involvement of chemokines and their corresponding receptors in pathogenesis of prostate cancer has gained tremendous attention. We have previously shown that CC chemokines receptor-9 (CCR9) is significantly expressed in prostate cancer cell lines compared to normal epithelial cells derived from prostate. Furthermore, we have shown higher invasive and migratory potential of prostate cancer cells under the chemotactic gradient of CCL25, the only known natural ligand of CCR9. The aim of this study was to establish the clinical relevance of CCR9 in prostate cancer using tissue micro array (TMA). The TMAs were stained for CCR9 and virtual slides were created using an Aperio ScanScope scanning system to numerically analyze the Immuno-intensity of CCR9. The ScanScope generated true color digital images of each stained sample, which were viewed and analyzed using Aperio ImageScope (version 6.25 software). Our data show higher expression of CCR9 in prostate cancer tissues compared to match benign tissues. Interestingly, CCR9 immuno-intensity was stronger in tissues with Gleason grade 8 and above. This study suggests the potential involvement of CCR9 in pathogenesis of prostate cancer.

Citation Format: Rajesh Singh, Pranav Gupta, William E. Grizzle, James W. Lillard, Shailesh Singh. Differential expression of CC chemokine receptor-9 in prostate cancer. [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 5121. doi:10.1158/1538-7445.AM2013-5121
Abstract 513: Breast cancers are notched up with a proton pump at their leading edge.
Authors: Pamarthy Sahithi , Gilman-Sachs Alice , Beaman Kenneth
Published: August 2014

The H+ vacuolar ATPase is a multimeric complex, which acidifies intracellular vesicles and extracellular space. Notch signaling is a key signaling pathway regulating cell proliferation and differentiation during development and cancer. The expression of both V-ATPases and Notch signaling is aberrant in most cancers and hence, are potential targets for therapeutics. It is speculated that the vacuolar ATPases regulate Notch signaling directly by endosomal acidification and indirectly by endocytosis. Here, we studied the role of the a2 isoform of vacuolar ATPase (a2V) in regulating Notch Signaling in Breast cancer. Using confocal microscopy, we traced the location of a2V in MCF7, SKBR3 and MDA-MB231 breast cancer cell lines. We found that a2V is present on the plasma membrane and not on the early endosomes. The amount of a2V correlated with the invasiveness of the cancer. Most importantly, the expression of a2V was restricted to one side of the cells marking their leading edge. Also, no a2V expression was found when cells contacted each other on the inside. Next, we evaluated the effect of a2V knockdown in MDA-MB231 cells. Using siRNA, a2V was knocked down. We found that decrease of a2V enhances the expression of Notch2 and Notch 4 significantly (>1.75 fold). Notch ligands (DLL family) and Notch targets also increased, supporting the finding. When we looked at Wnt pathway genes, we found a significant increase in Wnt4 (>2 fold) and c-Myc(∼3 fold) expression although levels of β-catenin were unchanged. Since c-Myc is known to mediate apoptosis, we looked at apoptotic genes Bax and Bcl2 to after siRNA knockdown of a2V. Although Bax increased, there was no significant change in apoptosis, as seen in PI-annexin assay. Taken together, our data suggests that a2V is important for tumor growth and its presence on leading edge marks aggressiveness of tumors. Interestingly, a2V depletion results in the activation of Notch and several proliferative markers in breast cancer. We postulate that this is due to indirect effects of a2V in endocytosis or in regulating pH dependent enzymes responsible for Notch and Wnt signal transduction.

Citation Format: Sahithi Pamarthy, Alice Gilman-Sachs, Kenneth Beaman. Breast cancers are notched up with a proton pump at their leading edge. [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 513. doi:10.1158/1538-7445.AM2013-513
Abstract 5139: Molecular docking exploration of potential RET tyrosine kinase inhibitors at non ATP-binding sites.
Authors: Nicolescu Adrian C. , Gujral Taranjit S. , Zelt Jordan DS , Mulligan Lois M.
Published: August 2014

RET (REarranged during Transfection) tyrosine kinase receptor is a transmembrane protein required for the development of neural-crest-derived cells, the urogenital system, and the central and peripheral nervous systems, notably the enteric nervous system. The RET protein has an extracellular domain with a cysteine-rich region and four cadherin-like domains, a transmembrane domain, and an intracellular tyrosine kinase domain, required for RET phosphorylation and downstream signaling. The structure of the RET kinase shares with other tyrosine kinases many conserved functional motifs and regulatory residues that are important for the kinase enzyme function. RET activation requires binding of a glial cell-line-derived neurotrophic factor (GDNF) and a co-receptor of the GDNF family receptors α. Germline mutations of RET, leading to uncontrolled activation, are associated with thyroid cancer, and recently with colorectal and lung cancers, and chronic myelomonocytic leukemia. RET mutations that result in decreased receptor function have been linked to developmental defects, such as Hirschsprung disease and kidney anomalies. The design of receptor tyrosine kinase (RTK) inhibitors has traditionally targeted the enzymes’ highly conserved ATP binding pocket. This approach resulted in the discovery of potent small molecule inhibitors, but with relatively low selectivity. To date there are no RET-specific inhibitors available for therapy, although few small molecule inhibitors are undergoing clinical evaluations as potential RET inhibitors. Since there are important critical regions within the RET molecule (e.g., the substrate binding pocket and activation loop) that contain unique amino acids and structural features, we used the method of molecular docking to virtually screen a diverse library of compounds that potentially target non-ATP binding sites of RET. Known ligands of RET were used to test the success of docking before the library of compounds was screened, docking solutions were inspected and ranked, and best compounds selected for novel structural features and future testing. Multiple comparisons with other known structures of RTK-ligand complexes have been performed in order to identify molecular features that would characterize the discovery of potential RET-specific inhibitors.

Citation Format: Adrian C. Nicolescu, Taranjit S. Gujral, Jordan DS Zelt, Lois M. Mulligan. Molecular docking exploration of potential RET tyrosine kinase inhibitors at non ATP-binding sites. [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 5139. doi:10.1158/1538-7445.AM2013-5139

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 5157: Ubiquitin-dependent regulation of AKT by NEDD4-1.
Authors: Wang Xinjiang , Fan Chuan-Dong , Lum Michelle A. , Xu Chao , Black Jennifer D.
Published: August 2014

Several E3 ligases have been reported to regulate AKT stability and activation through ubiquitination. We found that NEDD4-1 is a novel E3 ligase involved in ubiquitin-dependent regulation of AKT. NEDD4-1 physically interacts with AKT and mediates AKT ubiquitination in vitro and in vivo. NEDD4-1 catalyzes K63-type polyubiquitin chain formation on AKT in an in vitro ubiquitination assay. AKT ubiquitination by NEDD4-1 in vivo requires plasma membrane binding but not phosphorylation of AKT. Ubiquitination of AKT or pAKT by NEDD4-1 promotes AKT nucleus-oriented translocation. IGF-1 signaling stimulates NEDD4-1-mediated ubiquitination of pAKT but not total AKT. A cancer-derived plasma membrane-philic mutant AKT(E17K) is more effectively ubiquitinated by NEDD4-1 and more efficiently trafficked into the nucleus compared with AKT. This study revealed a novel function of AKT ubiquitination by NEDD4-1 in its nuclear trafficking, a step that contributes to AKT activation process in IGF-1 response.

Citation Format: Xinjiang Wang, Chuan-Dong Fan, Michelle A. Lum, Chao Xu, Jennifer D. Black. Ubiquitin-dependent regulation of AKT by NEDD4-1. [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 5157. doi:10.1158/1538-7445.AM2013-5157
Abstract 5165: Pathway oriented phospho-proteomics analysis with reverse phase protein array (RPPA) technology in cell growth and cancer therapy.
Authors: Inoue Takeomi , Iwata Naoko , Nishiwaki Eiji , Kirii Yasuyuki
Published: August 2014

A number of molecularly targeted drugs have been successfully launched as anti-cancer agents, and intensive efforts are underway to discover new targets and their modifiers. Nevertheless, many compounds selected by enzyme screening methodology demonstrated unexpected effects in intact cells or animal models. Since recent efforts from signaling pathway analysis have revealed that complexity of signaling networks, homeostatic controls and off-target effects can lead to unexpected cellular/in vivo consequences, profiling the effects of drug candidates on a wide range of intracellular signaling pathways is becoming an attractive strategy.

Many proteomics approaches, especially in the area of phospho-proteomics, have been developed, and their applications are rapidly growing for the purpose of cellular signal profiling. Among these approaches, reverse phase protein array (RPPA) is one of the most powerful technologies because of its high sensitivity and reliability. We have developed a signal transduction oriented RPPA system that focuses on phospho-protein profiles in samples. Thus far, our system utilizes approximately 180 phospho-protein specific antibodies and can profile the phosphorylation status of more than 100 Gene Ontology processes covering more than 10 components in each process. Our current studies on modification of cellular signals induced by tyrosine kinase inhibitors (TKIs) in several cancer cell types are elucidating novel mechanisms for TKI actions. For example, our studies on Erlotinib and Gefitinib treated cells have resulted in very similar profiles as EGF-stimulated cancer cells. However, several differences in the signaling profiles between them may suggest differences of efficacy and toxicity.

Independently, we have developed a nearly comprehensive in vitro kinase inhibitor profiling system, and we already have complete inhibition profiles for nine kinase inhibitors such as Erlotinib or Gefitinib. This allows us to combine in vitro profiling results with pathway intervention profiles to build a database integrating kinase-inhibition-potencies and cellular-signaling-profiles. Our aim is to expand this database to obtain new information to guide strategies for effective targeting in drug discovery. In this presentation, we present the novel targeting discovery strategy based on this database analysis.

Citation Format: Takeomi Inoue, Naoko Iwata, Eiji Nishiwaki, Yasuyuki Kirii. Pathway oriented phospho-proteomics analysis with reverse phase protein array (RPPA) technology in cell growth and cancer therapy. [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 5165. doi:10.1158/1538-7445.AM2013-5165
Abstract 5167: Profiling GTPases in native proteomes with a GTP acyl phosphate probe.
Authors: Nomanbhoy Tyzoon K. , Shih Ann , Aban Arwin
Published: August 2014

GTPases constitute an important class of regulatory proteins that participate as signal transducers in the capacity of molecular switches. The GTPase superfamily consists of 250 members in the human genome. These fall into several families, including the small GTPases (Ras Rho, Arf, Rab and Ran), heterotrimeric GTPases, elongation and initiation factors, and dynamins. In order to profile the distribution of GTPases in various cell lines and tissues, we have developed a chemical probe comprised of GTP linked to desthiobiotin through an acyl-phosphate bond. By treating complex proteomes with the probe, we are able to isolate probe-labeled peptides with streptavidin, and subsequently, identify the probe-labeled peptides by mass spectrometry. GTPases were identified from almost all known GTPase families indicating that this approach can be used to globally profile GTPases in various proteomes. The most common labeling site occurs at the conserved lysine residue in the phosphate binding loop (GXXXXGK) of the GTP-binding site. Significantly, the probe-labeled peptide for H-,K- and N-RAS includes amino acid positions 12 and 13, which are frequently mutated in cancers. Thus, the GTP probe can be used to directly quantify mutant RAS isoforms at the protein level.

Citation Format: Tyzoon K. Nomanbhoy, Ann Shih, Arwin Aban. Profiling GTPases in native proteomes with a GTP acyl phosphate probe. [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 5167. doi:10.1158/1538-7445.AM2013-5167
Abstract 5176: PPARγ acetylation governs differentiation function.
Authors: Tian Lifeng , Wang Chenguang , Hagen Fred K. , Casimiro Mathew , Sauve Anthony A. , Pestell Richard G.
Published: August 2014

The peroxisome proliferator-activator receptor gamma (PPARγ) is a member of nuclear receptors (NRs) that plays an important role in the pathogenesis of human diseases, including obesity, type II diabetes and cancer. PPARγ regulates lipid homeostasis by activating the transcription of key genes that are responsible for lipid biosynthesis. NRs undergo post-translational modification including phosphorylation and acetylation. Direct nuclear receptor acetylation by histone acetyltransferases regulates ligand sensitivity and growth properties. Using mass spectrometry and in vivo labeling assays we showed that PPARγ is acetylated in cultured cells. Inhibition of endogenous TSA-sensitive and NAD-dependent deacetylase activity enhanced Pparγ acetylation. SIRT1 bound and deacetylated Pparγ at K154/155. These lysine residues of Pparγ were required for adipocyte differentiation function and determined distinct gene expression signaling pathways. The lysine residues governing sumoylation (K77) and acetylation (K154/155) were shown to have dissociable functions in regulating cell-cycle, gene expression, gene promoter activity, and protein stability. Breast tumor growth, de novo lipid synthesis, induction of autophagy and evasion of apoptosis was promoted by K154/155 and inhibited by K77 in vivo. Pparγ acetylation induced a gene signature that was increased in breast cancer, associated with a reduction in SIRT1 abundance and poor outcome. These studies suggest that the Pparγ lysine motif regulates both ligand-dependent receptor stability and receptor-dependent gene expression and cell functions. Pparγ and SIRT1 signaling converge in vivo to regulate breast tumor lipid metabolism and growth.

Citation Format: Lifeng Tian, Chenguang Wang, Fred K. Hagen, Mathew Casimiro, Anthony A. Sauve, Richard G. Pestell. PPARγ acetylation governs differentiation function. [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 5176. doi:10.1158/1538-7445.AM2013-5176
Abstract 5177: SIRT2 deacetylates and activates glucose-6-phosphate dehydrogenase (G6PD) to protect cells against oxidative stress.
Authors: Wang Yiping , Zhou Lisha , Zhao Yuzheng , Wang Shiwen , Yang Yi , Ye Dan , Xiong Yue , Guan Kun-Liang
Published: August 2014

Glucose-6-phosphate dehydrogenase (G6PD) catalyzes the first committed step in the pentose phosphate pathway (PPP), which converts β-D-glucose-6-phosphate into D-glucono-1,5-lactone-6-phosphate, while concomitantly producing reducing equivalents in the form of NADPH. NADPH is a required cofactor in reductive biosynthesis,and is also required for the generation of reduced glutathione, the major antioxidant responsible for preventing reactive oxygen species (ROS) damage. Lysine acetylation is an evolutionarily conserved post-translational modification in the regulation of a wide range of cellular processes. Several recent acetylome proteomic studies have identified more than 2000 potential acetylation substrates. Among these identified acetylated proteins is G6PD, implicating a novel modulation of G6PD at the post-translational level. However, our knowledge of how acetylation regulates G6PD activity is limited. This study is directed toward to understand the molecular mechanism by which acetylation regulates G6PD function to regulate cellular redox state in response to oxidative stress. Here, we found that G6PD activity is negatively modulated by acetylation, and the major regulated site of acetylation in G6PD was mapped to K403, an evolutionarily conserved lysine residue. Site-specific genetic incorporation of Nε-acetyl-lysine into the position 403 of G6PD revealed that K403 acetylated G6PD is incapable to form active dimers and displays a complete loss in activity. Moreover, we found that the reversible acetylation of K403 is critical to modulate G6PD activity in response to different physiologic conditions, such as high glucose and oxidizing agents. Cytosolic SIRT2 deacetylated G6PD at K403, and greatly restored G6PD activity by inducing the formation of active dimers. Knockdown of endogenous G6PD led to higher cellular susceptibility to oxidant-induced cell death. Re-expression of wild-type G6PD, but not the acetylated mimic G6PDK403Q, could rescue cells from oxidative injury. Together these results uncover a previously unknown mechanism by which SIRT2 deacetylates and activates G6PD and thus protects cells against ROS damage.

Citation Format: Yiping Wang, Lisha Zhou, Yuzheng Zhao, Shiwen Wang, Yi Yang, Dan Ye, Yue Xiong, Kun-Liang Guan. SIRT2 deacetylates and activates glucose-6-phosphate dehydrogenase (G6PD) to protect cells against oxidative stress. [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 5177. doi:10.1158/1538-7445.AM2013-5177

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 5237: Systems level modeling following BRAF inhibition reveals an essential role for Ephrin A2 receptor in therapeutic escape.
Authors: Paraiso Kim H. T. , Fang Bin , Han Dale , Fedorenko Inna , John Jobin , Koomen John , Wargo Jennifer , Flaherty Keith , Meier Friedegund , Smalley Kerian S. M.
Published: August 2014

Although initially striking, the therapeutic efficacy of BRAF inhibition is eventually undermined by adaptive cellular signaling that leads to progressive disease. To date, several mechanisms of BRAF inhibitor resistance have been reported focusing primarily on single alterations. To address how global rewiring of melanoma signaling promotes resistance, we utilized mass spectrometry based phosphoproteomic and network analyses to identify signaling changes in tyrosine, threonine and serine that occur prior to and following acquired BRAF inhibitor resistance. This comprehensive bioinformatics approach uncovered a “resistance interactome” consisting of ∼150 nodes that was marked by significant alterations and the emergence of signaling nodes such as ITGB1, EphA2, EphB4, FAK1, STAT3 and PXN. GeneGo pathway enrichment analysis revealed enhanced cytoskeletal rearrangement, adhesion, integrin signaling, cell migration and extracellular matrix remodeling. Consistent with our bioinformatic predictions, melanoma cell lines with acquired vemurafenib resistance were considerably more invasive than their drug naïve counterparts and showed increased trans-endothelial cell migration. Confirmatory Western blot studies showed increased expression and phosphorylation of EphA2 receptor at Ser897, FAK1 at Ser397 and paxillin at Y118. Knockdown of EphA2 in the resistant cultures inhibited ERK signaling and completely prevented cell invasion and trans-endothelial cell migration. Functionally, EphA2 expression appeared to be regulated by an epigenetic mechanism involving promoter methylation and HDAC1. The clinical relevance of these findings was demonstrated in matched pairs of biopsies from patients receiving either BRAF or BRAF+MEK inhibitors, with significant Ephrin A2 receptor expression being observed in a subset of those on therapy. In summary, chronic MAPK pathway inhibition leads to major remodeling of the melanoma signaling network with EphA2 emerging as a key driver of the resistance phenotype.

Citation Format: Kim H. T. Paraiso, Bin Fang, Dale Han, Inna Fedorenko, Jobin John, John Koomen, Jennifer Wargo, Keith Flaherty, Friedegund Meier, Kerian S. M. Smalley. Systems level modeling following BRAF inhibition reveals an essential role for Ephrin A2 receptor in therapeutic escape. [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 5237. doi:10.1158/1538-7445.AM2013-5237
Abstract 524: Cyclooxygenase pathway in ovarian cancer.
Authors: Rao Gautam G. , Kochel Tyler , Kundu Namita , Reader Jocelyn , Fulton Amy
Published: August 2014

Objectives: To examine the role of the cyclooxygenase (COX) pathway in ovarian cancer. Cyclooxygenases, specifically COX1 and COX2, are frequently overexpressed in malignancies and lead to the synthesis of prostaglandins. A particularly important prostaglandin, PGE2 is exported from the cell into the extracellular milieu by the multi-drug resistance protein 4 (MRP4) transporter where it acts in a paracrine and autocrine manner by activating a family of G-protein coupled receptors (EP1-4). PGE2 is transported into the cell by the prostaglandin transporter (PGT) and catabolized by 15-prostaglandin dehydrogenase (15-PGDH). It has been previously shown that COX1, rather than COX2, is overexpressed in ovarian cancer. Little is known about the role of other members of this pathway, including EP receptors, PGT, MRP4, and 15-PGDH, in this disease.

Methods: HOSE (benign immortalized ovarian epithelial cells) and two ovarian cancer cell lines, SKOV3 and OVCAR3, were grown under standard conditions. Cells were harvested and RNA and protein were isolated. Quantitative RT-PCR was done using the SYBR Green protocol to examine the expression of members of the cyclooxygenase pathway and expressed relative to HOSE cells.

Results: In both ovarian cancer cell lines relative to HOSE, expression of COX2, PGT, EP4, and EP1 was decreased. In SKOV3 cells, COX1 and 15-PGDH expression was decreased but MRP4 levels were comparable to levels detected in HOSE cells. In OVCAR3 cells, COX1 and 15-PGDH expression levels were increased and MRP4 was decreased relative to HOSE cells. PGE2 levels in media will be measured to determine the net effect of these changes. Protein levels of the members of the cyclooxygenase pathway will be determined by western blot.

Conclusions: It is clear that several members of the cyclooxygenase pathway are expressed differently in malignant versus normal ovarian cells. Studies in progress will determine the functional significance of these differences.

Citation Format: Gautam G. Rao, Tyler Kochel, Namita Kundu, Jocelyn Reader, Amy Fulton. Cyclooxygenase pathway in ovarian cancer. [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 524. doi:10.1158/1538-7445.AM2013-524
Abstract 5242: Effect of total saponins of Gynostemma pentaphyllum on melanogenesis and anti-melanoma effect in B16 cells.
Authors: Tsang Ting Fung , Tai William Chi Shing , Hsiao Wendy Wen Luan
Published: August 2014

Melanogenesis is a physiological process of melanin production in response to UV exposure, which is modulated through multi-signaling pathways including cAMP/PKA, Wnt/β-catenin and MAPK signaling cascades. In this study, we investigated both the melanogenesis and anti-melanoma effects of total saponins of Gynostemma pentaphyllum (GpS). Our results showed that non-toxic dosages of GpS stimulated tyrosinase activity and increased melanin content in a dose-dependent manner. Western blot analysis showed that GpS treatment significantly up-regulated the expression levels of the melanogenic proteins, tyrosinase (TYR) and microphthalmia-associated transcription factor (MITF) in a dose-dependent manner. The p-CREB, which is the down-stream target of PKA is also elevated upon GpS treatment. We further observed that H89, a PKA inhibitor, attenuated the GpS induced tyrosinase activity, melanin content, and the protein expression of p-CREB and the nuclear β-catenin. To identify the active saponins, GpS was fractionated in the MCI-CHP 20P column and eluted with methanol in a gradient of 50 to 100% . The resulting ten fractions were tested for their melanogensis activities. We found that Fractions 5 to 9 showed the strongest effect in melanin synthesis in B16 cultures, while up-regulated the expression levels of TYR and MITF, while Fractions 7 to 9 increased nucleus β-catenin protein expression. Further purification of GpS is under way. Our early works showed that GpS exhibits strong anti-cancer effects in both in cellular and animal model. We were wonder whether GpS would exert anti-cancer effect against melanoma. To address this question, we tested and showed that GpS inhibited B16 cells migration in a dose-dependent manner. Meanwhile, GpS also increased G1/S ratio of the GpS-treated B16 cells, suggesting that GpS may act as G1 inhibitor. Our study seems to be in line with the recent reports that stimulation of melanogenesis might be associated with the anti-melanoma effect by decreasing proliferation and invasiveness of melanoma (Chien, et al.,2009). Based on the above results, we proposed that GpS might exert the anti-melanoma effect by stimulating the melanogenesis through the activation of cAMP/PKA and Wnt/β-catenin signaling pathways.

Citation Format: Ting Fung Tsang, William Chi Shing Tai, Wendy Wen Luan Hsiao. Effect of total saponins of Gynostemma pentaphyllum on melanogenesis and anti-melanoma effect in B16 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 5242. doi:10.1158/1538-7445.AM2013-5242