bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4043: The role of CD63/Timp1/β1 integrin complex in cell survival.
Authors: Pinto Mariana T. , de Melo Fabiana Henriques Machado , Jasiulionis Miriam Galvonas
Published: August 2014

Although malignant melanoma is the less frequently diagnosed skin cancer, it shows a poor prognosis due its chemoresistance and metastasis development. One of the acquired abilities of transformed cells is anoikis resistance and this property is closely related to metastasis formation. In our laboratory, we developed a model that allows us to study different steps of melanocyte malignant transformation. Melan-a melanocytes surviving after 1, 2, 3 and 4 deadhesion cycles showed modified morphology and independent PMA growth and have been named 1C, 2C, 3C and 4C cells, respectively. Different melanoma cell lines were established after submitting 4C spheroids to limiting dilution. Previous data from our group demonstrated increased expression of Timp1 and resistance to anoikis during the malignant transformation of melanocytes. Melan-a melanocytes overexpressing Timp1 acquire anoikis-resistant phenotype. In our laboratory, we observed interaction between CD63, Timp1 and β1-integrin in the 4C, 4C11- and 4C11+ cell lines established after cycles of anchorage impediment. This seems to be the first study describing the interaction between Timp1, CD63 and β1-integrin in tumor cells. However, it is unclear how Timp1 modulates resistance to anoikis. Therefore, our aim is to analyze the role of Timp1 in the context of Timp1/CD63/β1-integrin complex and the signaling pathways involved in the acquisition of anoikis resistance. Supported by CAPES and FAPESP.

Citation Format: Mariana T. Pinto, Fabiana Henriques Machado de Melo, Miriam Galvonas Jasiulionis. The role of CD63/Timp1/β1 integrin complex in cell survival. [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 4043. doi:10.1158/1538-7445.AM2013-4043
Abstract 4046: Estrogen receptor-α coordinates the unfolded protein response,autophagy, and reactive oxygen species generation to regulate breast cancer survival.
Authors: Clarke Pamela A. , Cook Katherine , Hu Rong , Schwartz Jessica L. , Abu-Asab Mones , Warri Anni , Clarke Robert
Published: August 2014

Of the almost 240,000 new breast cancers diagnosed in 2011 within the USA, approximately 70% of these cases express the estrogen receptor-α (ER). ER targeted therapies such as tamoxifen (TAM) or faslodex (ICI) are often used to treat this breast cancer subtype. However, the curative potential of these interventions is frequently limited in patients due to resistance. Understanding how antiestrogen resistance occurs could lead to improved approaches to eradicate ER+ breast cancers. Increased unfolded protein response (UPR, an endoplasmic reticulum stress pathway) or autophagy (a “self” eating process used to clear dysfunctional protein or organelles) was previously shown to promote antiestrogen resistance in ER+ breast cancer. Using the antiestrogen sensitive MCF7-LCC1 (LCC1) and resistant MCF7-LCC9 (LCC9) cell lines, we determined the effect of antiestrogens and ERα on autophagy and UPR signaling. Suprisingly, ablation of ERα through RNAi potentiated LCC1 to antiestrogen-mediated cell death and resensitized LCC9 cells to endocrine therapy. While ICI stimulated both autophagy and UPR signaling, RNAi knockdown of ERα in LCC1 and LCC9 cells inhibited ICI-mediated UPR signaling and concurrently stimulated autophagy. Furthermore, we showed that ERα knockdown stimulated ROS production and potentiated the cell death response to antiestrogen therapy. ERα knockdown, but not ICI treatment, reduced nuclear Nrf2 (a UPR-induced antioxidant signaling protein) and increased cytosolic KEAP1 (an inhibitor of Nrf2), which may lead to the observed increase in ROS production. Autophagy inhibition through ATG7 silencing increases cell death and apoptosis in LCC1 ERα knockdown cells, showing that autophagy induction by antiestrogens is prosurvival in this context. These data suggests that UPR and autophagy stimulated by antiestrogen therapy results from two different mechanisms of ER regulation. We show that antiestrogens promote the accumulation of ERα in the cytosol that is associated with increased UPR signaling, while the inhibition of ERα promotes prosurvival autophagy. The inherent stimulation of resistance mechanism signaling through antiestrogen treatment suggests that combining autophagy or UPR inhibitors with anti-estrogens may greatly reduce the development of acquired antiestrogen resistance in breast cancer.

Citation Format: Pamela A. Clarke, Katherine Cook, Rong Hu, Jessica L. Schwartz, Mones Abu-Asab, Anni Warri, Robert Clarke. Estrogen receptor-α coordinates the unfolded protein response,autophagy, and reactive oxygen species generation to regulate breast cancer survival. [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 4046. doi:10.1158/1538-7445.AM2013-4046
Abstract 4048: Aggresome formation is regulated by RanBPM through an interaction with HDAC6.
Authors: Salemi Louisa , Almawi Ahmad , Lefebvre Karen , Schild Poulter Caroline
Published: August 2014

RanBPM (Ran-binding protein M, also called RanBP9) is a nucleocytoplamsic protein that has been implicated in a number of cellular processes including cell adhesion, migration and gene transcription. It has been hypothesized to be a scaffolding protein, but its function remains poorly understood. In previous studies we documented the relocalization of RanBPM from the nucleus to the cytoplasm in response to ionizing radiation (IR) treatment. Subsequent analysis showed that IR triggered cytoplasmic RanBPM relocalization in perinuclear dots or foci often located close to an invagination of the nucleus. Upon further investigation, the RanBPM aggregates were identified as aggresomes, which have not been previously documented to occur in response to IR. Aggresomes are aggregates of misfolded/ubiquitinated proteins resulting from improper protein assembly, aberrant modifications and/or environmental stress. These misfolded/ubiquitinated proteins are normally cleared by chaperone-mediated refolding or by proteasomal degradation through the ubiquitin-proteasome system. In conditions where these systems are impaired or overwhelmed, misfolded proteins accumulate in specific perinuclear structures called aggresome. Further analysis confirmed that the RanBPM foci were indeed aggresomes by assessing co-localization of RanBPM with HDAC6, which is required for aggresome formation. Further, we showed RanBPM also formed aggresome in response to treatment with the proteasome inhibitor MG132. To determine the effect of RanBPM downregulation on aggresome formation, we assessed aggresome formation by HDAC6 in RanBPM shRNA and control shRNA Hela and HEK cells. RanBPM downregulation resulted in significantly lower number of aggresomes both after MG132 and IR treatment, suggesting that RanBPM is essential in aggresome formation. Confocal microscopy analysis showed that RanBPM and HDAC6 co-localize within the aggresome and co-immunoprecipitation studies detected an interaction between the two proteins. Downregulation of RanBPM also resulted in decreased acetylated α-tubulin, suggesting that RanBPM regulates HDAC6 activity. Altogether these results suggest that RanBPM may regulate aggresome formation through an interaction with HDAC6. The molecular details of this regulation are currently under investigation.

Citation Format: Louisa Salemi, Ahmad Almawi, Karen Lefebvre, Caroline Schild Poulter. Aggresome formation is regulated by RanBPM through an interaction with HDAC6. [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 4048. doi:10.1158/1538-7445.AM2013-4048
Abstract 4070: β-arrestin-2 mediated regulation of plasminogen activator inhibitor Type 1.
Authors: Sobolesky Philip M. , Zhou Tanyau , Gorstein Arielle , Woolworth Julie A. , Moussa Omar
Published: August 2014

Several studies described the elevated levels of plasminogen activated Inhibitor-1 (PAI-1) protein in malignant tissues and indicated the potential use as a therapeutic target. In bladder cancer, higher levels of PAI-1 were found in tumor tissues compared to normal tissues. Urinary levels of PAI-1 protein in combination with other markers resulted in higher sensitivity and specificity in detecting bladder cancer. In human kidney mesangial cells, the thromboxane A2 (TP) receptor agonist stimulation increased PAI-1 mRNA and protein. Our lab indicated that TP receptor is highly expressed in bladder cancer tissues and inversely correlated with patients` survival. We also indicated the TPβ isoform can induce malignant transformation in immortalized non-transformed urothelial cells. Our published data indicated that U46619 dependent stimulation of cell migration is β-arrestin-2 dependent. The purpose of the current study is to investigate the mechanism whereby TP stimulation regulates PAI-1 expression. The protein levels of PAI-1 were found to be increased in bladder cancer cells following stimulation with TP agonist (U46619). Tetracycline inducible shRNA knock-down of β-arrestin-2 resulted in a significant reduction of PAI-1 protein, but not mRNA in bladder cancer cells and also resulted in a significant reduction of trans-well cell migration and invasion. Our data suggest that the agonist mediated increase in PAI-1 is regulated at the post-transcriptional level. The role of β-arrestin-2 KD on agonist dependent stimulation remains to be determined.

Citation Format: Philip M. Sobolesky, Tanyau Zhou, Arielle Gorstein, Julie A. Woolworth, Omar Moussa. β-arrestin-2 mediated regulation of plasminogen activator inhibitor Type 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 4070. doi:10.1158/1538-7445.AM2013-4070
Abstract 4073: Proteasomal degradation of JunD is required for TGF-β induced inhibition of proliferation in prostate cancer cells.
Authors: Millena Ana C. , Vo BaoHan T. , Strong Nicole , Walker Lindsey , Cao Yang , Klueva Natalya , Pfarr Curt , Khan Shafiq
Published: August 2014

Transforming growth factor-β (TGF-β) inhibits proliferation of prostate epithelial cells. However, prostate cancer cells in advanced stages become resistant to TGF-β effects on proliferation. In this study, we have investigated the role of AP-1 family of transcription factors, specifically the Jun family members (c-Jun, JunB and JunD) in TGF-β1 effects on prostate cancer cell proliferation. Basal expression of individual Jun family members was determined by RT-PCR and Western blot analysis in four prostate cell lines: RWPE1, LNCaP, DU145 and PC3 cells. Jun levels were also examined in response to TGF-β1. c-Jun was expressed in all cell lines and its expression was up-regulated by TGF-β1. Levels of JunB were higher in normal cells compared to those in cancer cells and TGF-β1caused an increase in JunB levels. JunD levels were higher in all cell lines; TGF-β1 had differential effects on JunD protein levels. While it caused a significant decrease in JunD levels in RWPE-1 and DU145 cells, it did not affect JunD levels in PC3 cells. These differential effects on JunD levels correlated with differential effects of TGF-β on cell proliferation. Individual Jun family members were overexpressed in DU145 cells and effects on cell proliferation were determined. Over-expression of c-Jun and JunB decreased proliferation rate in DU145 cells; however, overexpression of JunD increased proliferation in these cells. Interestingly, cells over-expressing JunD still succumb to inhibitory effect of TGF-β1 after six to eight days of incubation. Furthermore, silencing JunD by siRNA decreased proliferation in both DU145 and PC3 cells. To determine the molecular mechanism of TGF-β-induced down-regulation of JunD, we pretreated cells over-expressing JunD with a proteosomal inhibitor, MG132. Pretreatment of MG132 blocked the degradation of JunD in in DU145 cells over-expressing JunD. In conclusion, our studies show that specific Jun family exerts differential effects on proliferation in prostate cancer cells in response to TGF-β1. While c-Jun and Jun B mediate the inhibitory effects of TGF-β1 on proliferation, JunD counteracts these effects. TGF-β1 causes inhibition in proliferation by degrading JunD via proteasomal degradation.

Acknowledgements: These studies were supported by the NIH/NIMHD/RCMI grant #G12MD007590, NIH/NIMHD #5P20MD002285, and DOD grant # W8I-08-1-0077.

Citation Format: Ana C. Millena, BaoHan T. Vo, Nicole Strong, Lindsey Walker, Yang Cao, Natalya Klueva, Curt Pfarr, Shafiq Khan. Proteasomal degradation of JunD is required for TGF-β induced inhibition of proliferation in prostate 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 4073. doi:10.1158/1538-7445.AM2013-4073
Abstract 4074: Transcription factors involved in the genesis and progression of cancer differently modulated by transforming growth factor-beta3 (TGF-Beta3) in prostate cell lines.
Authors: Caggia Silvia , Candido Saverio , Libra Massimo , Cardile Venera
Published: August 2014

Transforming growth factor-beta3 (TGF-β3) is a member of a family of multifunctional cytokines that control different cellular processes including cell proliferation, morphogenesis, migration, extracellular matrix production, cytokine secretion, and apoptosis, as well as in normal physiological and disease processes. Among the various isoforms of TGF-β, TGF-β1 has been the most studied form, especially in the context of tumor development. In contrast, only few studies have been focused on the role of TGF-β3 in tumorogenesis. Recently, it has been shown that the transcription factors Yin Yang 1 (YY1) is involved in the TGF-β3 pathway. YY1 is known to be associated with cancer progression. One the potential mechanisms of this association is linked with the inactivation of p53. On this ground, the aims of this study were i) to explore the correlated expression of p53 and YY1 in a human benign prostate hyperplasia cell line (BPH-1) and in two prostate cancer cell lines, LNCaP (androgen-sensitive) and DU-145 (androgen-refractory) after treatment with different dose of TGF-β3; ii) to determine, in TGF-β3-treated prostate cell lines, the expression of a fraction of intracellular signalling pathways involved in both cell cycle progression and in apoptosis. TGF-β3 stimulation in prostate cancer cell lines showed different effects, such as YYI downregulation and p53 overexpression. These effects are also associated with a different modulation of intracellular signaling pathways (PI3K, AKT, pAKT, PTEN, Bcl-2, Bax, and iNOS). In conclusion, these findings may have important therapeutic implications in prostate cancer.

Citation Format: Silvia Caggia, Saverio Candido, Massimo Libra, Venera Cardile. Transcription factors involved in the genesis and progression of cancer differently modulated by transforming growth factor-beta3 (TGF-Beta3) in prostate 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 4074. doi:10.1158/1538-7445.AM2013-4074
Abstract 4079: Control of survivin expression by IGF-I in prostate epithelial cells.
Authors: Danielpour David , Song Kyung , Shankar Eswar , Yang Jiayi , Bane Kara L.
Published: August 2014

Survivin, a unique member of the inhibitor of apoptosis (IAP) proteins, is overexpressed in numerous cancers through poorly defined mechanisms. We show that insulin-like growth factor-I (IGF-I), which is implicated in the etiology of prostate cancer, induces the expression of Survivin in a variety of pre-neoplastic and malignant prostate cell lines. Using the pre-neoplastic NRP-152 rat prostate cell line as a model, we showed that silencing Survivin by lentiviral-mediated shRNA represses IGF-I-stimulated cell growth, suggesting that the induction of Survivin is critical to this growth response. Moreover, we show that IGF-I induces the expression of Survivin through a transcriptional mechanism that is mediated in part by the PI3K/Akt/mTORC1 pathway. The CDE and CHR response elements in the proximal region of the Survivin promoter are decisive for this IGF-I response. TGF-β signaling antagonists similarly induced this promoter activity and rendered cells refractory to further promoter activation by IGF-I. Suppression of TGF-β signaling, either by TGF-β receptor kinase inhibitors or by silencing Smads 2 and 3, induced Survivin expression and promoted cell growth similar to that induced by IGF-I. TGF-β receptor antagonists also rescued cells from down-regulation of Survivin expression and growth suppression by pharmacological inhibitors of PI3K, Akt, MEK and mTORC1. Sh-RNA gene silencing studies suggest that mTORC1 induces while mTORC2 represses the expression of Survivin. We conclude that IGF-I signaling through a PI3K/Akt/mTORC1-dependent mechanism elevates expression of Survivin and promotes growth of prostate epithelial cells by suppressing Smad-dependent autocrine TGF-β signaling.

Citation Format: David Danielpour, Kyung Song, Eswar Shankar, Jiayi Yang, Kara L. Bane. Control of survivin expression by IGF-I in prostate epithelial 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 4079. doi:10.1158/1538-7445.AM2013-4079
Abstract 408: Hematogenous metastasis in ovarian cancer: Importance of the ErbB3/NRG axis.
Authors: Pradeep Sunila , Wook Kim , Nishimura Masato , Raghavan Pradeep , Pecot Chad , Lopez Gabriel , Fidler Isaiah , Sood Anil
Published: August 2014

Background: Ovarian cancer is predominantly thought to metastasize by intraperitoneal spread. Here, we report unexpected findings related to hematogenous ovarian cancer metastasis with strong tropism for the omentum.

Study methods and results: To investigate the potential patterns of metastasis from circulating ovarian cancer cells, we established a novel parabiosis model, where 2 mice are surgically fused to allow a common circulation. Using this model, we found that ovarian cancer cells can indeed metastasize hematogenously and predominantly localize to the omentum. Cells propagated from this model are called SKOV3-OM3. To understand potential mechanisms, we carried out integrated genomic and human Phospho RTK arrays, which pointed to ErbB3/NRG1 as a dominant activated pathway. NRG1 stimulated migration (40%) and invasion (60%) of SKOV3-OM3 cells. NRG1 stimulation consistently induced downstream signaling pathways such as PI3K-AKT axis and Src activation in the cells with predilection for omental metastasis. We also showed that EMT modulating genes (eg. Vimentin and E-cadherin) are regulated by ErbB3 and PI3K in SKOV3-OM3 cells. In parallel approaches to determine the requirement of the ErbB3/NRG1 pathway for hematogenous metastasis of ovarian cancer to the omentum, both immune-deficient and immune-competent models were used. Upon intracardiac (IC) injection of SKOV3-OM3 cells (nude mice) and IG10-OM2 cells (C57BL/6 mice), tumor development and metastasis was followed by weekly luciferase imaging; tumors were harvested for histopathology and downstream molecular analyses. ErbB3 silencing using siRNA incorporated in DOPC nanoliposomes resulted in substantially decreased tumor growth and metastasis compared with control siRNA in both models (P <0.001). ErbB3 silencing resulted in significant reduction of proliferation and increase in apoptosis for both experiments. These data indicate that ErbB3 plays a functionally significant role in omental metastasis.

Conclusion: This study provides a new paradigm regarding ovarian cancer metastasis in that ovarian cancer cells have a parallel path of spread to the omentum hematogenously. As such, these findings have significant clinical implications.

Citation Format: Sunila Pradeep, Kim Wook, Masato Nishimura, Pradeep Raghavan, Chad Pecot, Gabriel Lopez, Isaiah Fidler, Anil Sood. Hematogenous metastasis in ovarian cancer: Importance of the ErbB3/NRG axis. [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 408. doi:10.1158/1538-7445.AM2013-408
Abstract 4080: TGF-β Effects on prostate cancer cell migration are mediated by PGE2 through activation of PI3K/AKT/mTOR pathway.
Authors: Vo Baohan T. , Morton Derrick , Komaragiri Shravan , Millena Ana C. , Khan Shafiq A.
Published: August 2014

Transforming growth factor-beta (TGF-β) plays an important role in the progression of prostate cancer. It exhibits both tumor suppressor and tumor promoting activities. Correlations between COX-2 overexpression and enhanced production of PGE2 have been implicated in cancer progression; however, there are no studies indicating that TGF-β effects in prostate cancer cells involve PGE2 synthesis. In this study, we investigated TGF-β regulation of COX-1 and COX-2 expression in prostate cancer cells and whether the effects of TGF-β on cell proliferation and migration are mediated by PGE2. COX-1 protein was ubiquitously expressed in prostate cells; however, COX-2 protein levels were only detected in prostate cancer cells. TGF-β treatment increased COX-2 protein levels and PGE2 secretion in PC3 cells. Exogenous PGE2 and PGF2α had no effects on cell proliferation in LNCaP, DU145, and PC3 cells while PGE2 and TGF-β induced migratory behavior only in PC3 cells. Only EP2 and EP4 receptors were detected at mRNA levels in prostate cells. The EP4-targeting siRNA inhibited PGE2 and TGF-β-induced migration of PC3 cells. PGE2 and TGF-β induced phosphorylation of AKT which was blocked by antagonists of PGE2 (EP4) receptors (L161982, AH23848) and PI3-kinase inhibitor (LY294002) in PC3 cells. Pretreatment with L161982 or AH23848 blocked the stimulatory effects of PGE2 and TGF-β on cell migration, while LY294002 or rapamycin completely eliminated PGE2, TGF-β, and EGF-induced migration PC3 cells. We conclude that TGF-β increases COX-2 levels and PGE2 secretion in prostate cancer cells which, in turn, mediates TGF-β effects on cell migration through the activation of PI3K/AKT/mTOR pathway.

Acknowledgements: These studies were supported by the NIH/NIMHD/RCMI grant #G12MD007590, NIH/NIMHD #5P20MD002285, and DOD grant # W8I-08-1-0077.

Citation Format: Baohan T. Vo, Derrick Morton, Shravan Komaragiri, Ana C. Millena, Shafiq A. Khan. TGF-β Effects on prostate cancer cell migration are mediated by PGE2 through activation of PI3K/AKT/mTOR 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 4080. doi:10.1158/1538-7445.AM2013-4080
Abstract 4096: FAK inhibition with small molecule inhibitor Y15 decreases viability in thyroid cancer cell lines.
Authors: O'Brien Shalana B. , Golubovskaya Vita M. , Cance William G.
Published: August 2014

Introduction: Thyroid cancer is the most common endocrine malignancy and papillary the most common subtype. While low stage papillary carcinomas are treatable, for nonresectable tumors that do not respond to iodine, a treatment is still needed. In comparison, for advanced medullary cancer, a tyrosine kinase inhibitor, Vandetanib, is currently being used. Focal Adhesion Kinase (FAK) is a tyrosine kinase involved in many protein-protein interactions that control important cellular functions. FAK is upregulated in many cancer cell lines, including thyroid. It contains an autophosphorylation site Y397, which is blocked by 1,2,4,5-Benzenetetraamine tetrahydrochloride (Y15), a small molecule FAK inhibitor. We propose that this inhibitor, Y15, will decrease viability of thyroid cancer cell lines.

Methods: Four human thyroid cancer cell lines were cultured in vitro. K1, BCPAP, and TPC1 were derived from papillary thyroid carcinomas, and TT was derived from a medullary thyroid carcinoma. Western blots were used to detect expression of FAK and pY397 FAK in each untreated line. These were repeated with differing concentrations of Y15 plus the Novartis FAK inhibitor TAE226 as a positive control. MTS assays of each cell line were used to measure cell viability with increasing doses of Y15. Untreated medium, DMSO, and TAE226 were used as negative and positive controls.

Results: All untreated thyroid cancer cell lines expressed FAK and phosphorylated Y397 FAK on Western blotting. Increasing doses of Y15 in Western blotting decreased the expression of pY397 FAK in all cell lines. This was also associated with increased cell detachment. In MTS assays, viability was also decreased with Y15 in a dose-dependent manner across all cell lines. Significant inhibition was reached at relatively low doses: there was a 90% decrease in viability with 10μM Y15 in TPC1 cells and 5μM Y15 in TT cells. The effects were seen across all lines, but most profound in TPC1 and TT.

Conclusions: Papillary thyroid cancer cell lines and medullary carcinoma cells express FAK and pY397. The FAK inhibitor Y15 causes a dose-dependent decrease of phosphorylated FAK, and this disruption decreases cell viability. Our data suggests Y15 may be an effective potential treatment for advanced papillary thyroid cancer.

Citation Format: Shalana B. O'Brien, Vita M. Golubovskaya, William G. Cance. FAK inhibition with small molecule inhibitor Y15 decreases viability in thyroid 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 4096. doi:10.1158/1538-7445.AM2013-4096
Abstract 4100: YM-155 sensitizes malignant human glioma cells to ABT-737 via survivin and Mcl-1 downregulation in an EGFR-dependent context.
Authors: Jane Esther P. , Premkumar Daniel , DiDomenico Joseph , Hu Bo , Cheng Shi-Yuan , Pollack Ian
Published: August 2014

Antiapoptotic proteins are commonly overexpressed in gliomas, contributing to therapeutic resistance. We recently showed that clinically achievable concentrations of the Bcl-2/Bcl-xL inhibitor ABT-737 failed to induce apoptosis in glioma cells, with persistent expression of survivin and Mcl-1. To address the role of these mediators in glioma apoptosis resistance, we analyzed the effects of YM-155, a survivin suppressant, on survival on a panel of glioma cell lines. YM-155 inhibited growth, and downregulated survivin and Mcl-1 in a dose- and cell line-dependent manner. Whereas U373, LN18, LNZ428, T98G, LN229, and LNZ308 cells exhibited an IC50 of 10-75 nM, A172 cells were resistant (IC50 ∼ 250 nM). No correlation was found between sensitivity to YM-155 and baseline expression of survivin or cIAP-1/cIAP-2/XIAP. However, strong correlation was observed between EGFR activation levels and YM-155 response, which was confirmed using EGFR-transduced versus wild-type cells. Because we postulated that decreasing Mcl-1 expression may enhance glioma sensitivity to ABT-737, we examined whether cotreatment with YM-155 promoted ABT-737 efficacy. YM-155 synergistically enhanced ABT-737-induced cytotoxicity and caspase-dependent apoptosis. Down-regulation of Mcl-1 using shRNA also enhanced ABT-737-inducing killing, confirming an important role for Mcl-1 in mediating synergism between ABT-737 and YM-155. As with YM-155 alone, sensitivity to YM-155 and ABT-737

Citation Format: Esther P. Jane, Daniel Premkumar, Joseph DiDomenico, Bo Hu, Shi-Yuan Cheng, Ian Pollack. YM-155 sensitizes malignant human glioma cells to ABT-737 via survivin and Mcl-1 downregulation in an EGFR-dependent context. [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 4100. doi:10.1158/1538-7445.AM2013-4100
Abstract 4102: FAT1: A novel regulator of cancer and inflammation.
Authors: Dikshit Bhawana , Chattopadhyay Parthaprasad , Sinha Subrata , Chosdol Kunzang
Published: August 2014

FAT1, a member of cadherin superfamily is a large trans-membrane protein with 34 cadherin repeats. FAT1 was originally identified as a tumor suppressor gene in Drosophila but its role in human cancers is just beginning to unfold. Considering its tumor suppressive role in Drosophila, few studies have reported similar function of FAT1 in human cancers but on the contrary several studies have also demonstrated oncogenic role of FAT1. In this study we identified oncogenic role of FAT1 in human glioma where knockdown of FAT1 was found to inhibit migration and invasion of glial cells. Further investigation into molecular mechanism of FAT1 function revealed direct link to AP-1 mediated transcription via Programmed Cell Death 4 (PDCD4). Knockdown of FAT1 was found to inhibit AP-1 mediated transcription and expression of downstream target genes. AP-1 is a known mediator of cancer and inflammation and accordingly we found decreased expression of several pro-inflammatory markers like COX-2, cytokines like IL-6, IL-1β and chemokines like IL-8, MCP-1 and MIP-2 after FAT1 knockdown. Interestingly, the expression of anti-inflammatory cytokine IL-10 was found to be increased after FAT1 knockdown. Moreover FAT1 expression was also found to positively correalate with COX-2 and IL-6 expression in primary GBM tumors. Thus, in this study FAT1 was found to act as an oncogene as well as to orchestrate the inflammatory milieu of the glioma cells by regulating AP-1 mediated transcription via PDCD4.

Citation Format: Bhawana Dikshit, Parthaprasad Chattopadhyay, Subrata Sinha, Kunzang Chosdol. FAT1: A novel regulator of cancer and inflammation. [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 4102. doi:10.1158/1538-7445.AM2013-4102
Abstract 4104: Netrin-1 promotes the invasiveness and survival of human glioblastoma cells.
Authors: Keski-Oja Jorma K. , Ylivinkka Irene , Hu Yizhou , Nyman Tuula , Hyytiainen Marko
Published: August 2014

Glioblastoma multiforme (GBM) is the most severe human brain cancer, which has no efficient treatment. The main reason for the lethality of GBM is its resistance to all current therapies and highly invasive. According to microarray data on Oncomine database netrin-1 is upregulated in glioblastoma tumors. Netrin-1 is a secreted laminin related extracellular matrix protein. Its main function is to regulate the axon guidance during embryogenesis. Recently it has been observed to act as a survival factor for different forms of cancer such as aggressive neuroblastoma, metastatic breast cancer and non small cell lung cancer. It also promotes tumorigenesis of colorectal cancer.

Based on these observations we explored the possibility whether netrin-1 plays a role in human GBM. We analyzed the effects of netrin-1 expression on human GBM cells. According to Matrigel invasion assays overexpression of netrin-1 increased the invasiveness whereas partial knock-down of netrin-1 by shRNAs reduced the invasiveness of human GBM cells. On the other hand, complete knock-down of netrin-1 led to apoptotic death of those cells. To further analyze netrin-1 signaling in human GBM we performed a mass spectrometry screen to identify binding partners of netrin-1. We identified Notch as a novel receptor for netrin-1. In addition, we observed that netrin-1 overexpression increased and knockdown decreased Notch activation. The role of Notch signaling regarding the aggressiveness of various cancers has been controversial. Our results confirm that increased Notch signaling increases glioblastoma cell invasiveness, and that netrin-1 increases Notch signaling. Further, we identified here a recombinant netrin-1 fragment that inhibits Notch signaling by blocking the Notch signaling complex to the cell surface.

Current studies provide new mechanistic information on the tumorigenesis of GBM. Activation of Notch signaling pathway has been linked to the initiation of EMT which is essential for cancer cell invasion. Our results suggest that netrin-1 regulates GBM invasion by activating Notch signaling. The inhibition of netrin-1 signaling may prove out to be a treatment option for human glioblastoma.

Citation Format: Jorma K. Keski-Oja, Irene Ylivinkka, Yizhou Hu, Tuula Nyman, Marko Hyytiainen. Netrin-1 promotes the invasiveness and survival of human glioblastoma 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 4104. doi:10.1158/1538-7445.AM2013-4104
Abstract 4114: Trimeric G protein-CARMA1 axis links smoothened to NF-κB activation in diffuse large B-cell lymphoma.
Authors: Qu Changju , Liu Yadong , Kunkalla Kranthi , Singh Rajesh , Blonska Marzenna , Agarwal Nitin , Lin Xin , Vega Francisco
Published: August 2014

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoid malignancy in adults. Aberrant activation of Hedgehog (Hh) and NF-κB pathways is ubiquitously observed and known to mediate tumor growth, survival and chemo-resistance in DLBCL. Previously, we found that activation of Hh signaling is positively correlated with NF-κB pathway in DLBCL tumors and that modulating the activity of smoothened (SMO), the signal transducer subunit of Hh pathway, but not GLI1, transcriptional factor of Hh signaling, resulted in modulation of NF-kB pathway activation. Hereby, we investigate the molecular mechanisms that link SMO to NF-kB pathway. To elucidate whether there was interaction between G protein superfamily and SMO, we immunoprecipitated representative members of the Gα subfamilies (Gαi, Gα16, Gαq, Gαs and Gα12), and found that SMO was associated with Gαi, Gαq, and Gα12 in two DLBCL cell lines. Stimulation of SMO with Shh recombinant increased the recruitment of Gαi and Gα12 to SMO and also increased the GTPase activity of Gαi and Gα12. We also found that the activation of SMO with Shh in DLBCL cell lines was associated with increased total activity of PKC, phosphorylation of PKCβ1 and -2, and recruitment of CARMA1-MALT1-BCL10-TRAF6 to SMO receptor complex supporting that G proteins are involved in the transmission of the signal between SMO and NF-κB. Opposite results were seen when SMO was inhibited or silenced. As polyubiquitination of TRAF6 and NEMO (IKKγ) at lysine 63 (K63) are also important events in propagating NF-κB signaling, we examined the effect of SMO overexpression on K63-linked polyubiquitination of TRAF6 and NEMO. Transient overexpressing SMO resulted in increased polyubiquitination of TRAF6 and NEMO, supporting activation of both proteins by SMO. Moreover, functional inhibition of SMO (cyclopamine-KAAD) enhances the cytotoxic effect of NF-κB inhibitor (BAY-11-7082). Altogether, our study reveal a non-canonical Hh signaling pathway, in which SMO activates trimeric G proteins and CARMA1-associated signaling complex, leading to NF-κB activation. This signaling cascade contributes to the survival of DLBCL, and may serve as potential targets for combination therapies in DLBCL.

Citation Format: Changju Qu, Yadong Liu, Kranthi Kunkalla, Rajesh Singh, Marzenna Blonska, Nitin Agarwal, Xin Lin, Francisco Vega. Trimeric G protein-CARMA1 axis links smoothened to NF-κB activation in diffuse large B-cell lymphoma. [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 4114. doi:10.1158/1538-7445.AM2013-4114
Abstract 413: Overexpression of Snail is associated with lymph node metastasis and poor prognosis in patients with gastric cancer.
Authors: Kwon Chae Hwa , Shin Na Ri , Jeong Eun Hui , Choi Chang In , Moon Hyun Jung , Chu In Sun , Kim Gwang Ha , Jeon Tae Yong , Lee Jae Hyuk , Park Do Youn
Published: August 2014

Background: Epithelial-mesenchymal transition (EMT) plays a significant role in tumor progression and invasion. Snail is a known regulator of EMT in various malignant tumors. This study investigated the role of Snail in gastric cancer.

Methods: We examined the effects of silenced or overexpressed Snail using lenti-viral constructs in gastric cancer cells. Immunohistochemical analysis of tissue microarrays from 314 patients with gastric adenocarcinoma (GC) was used to determine Snail's clinicopathological and prognostic significance. Differential gene expression in 45 GC specimens with Snail overexpression was investigated using cDNA microarray analysis.

Results: Silencing of Snail by shRNA decreased invasion and migration in GC cell lines. Conversely, Snail overexpression increased invasion and migration of gastric cancer cells, in line with increased VEGF and MMP11. Snail overexpression (≥75% positive nuclear staining) was also significantly associated with tumor progression (P < 0.001), lymph node metastases (P = 0.002), lymphovascular invasion (P = 0.002), and perineural invasion (P = 0.002) in the 314 GC patients, and with shorter survival (P = 0.023). cDNA microarray analysis revealed 213 differentially expressed genes in GC tissues with Snail overexpression, including genes related to metastasis and invasion.

Conclusion: Snail significantly affects invasiveness/migratory ability of GCs, and may also be used as a predictive biomarker for prognosis or aggressiveness of GCs.

Citation Format: Chae Hwa Kwon, Na Ri Shin, Eun Hui Jeong, Chang In Choi, Hyun Jung Moon, In Sun Chu, Gwang Ha Kim, Tae Yong Jeon, Jae Hyuk Lee, Do Youn Park. Overexpression of Snail is associated with lymph node metastasis and poor prognosis in patients with gastric 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 413. doi:10.1158/1538-7445.AM2013-413
Abstract 4134: Development of MUT-MAP, a high-throughput multi-analyte mutation panel for cancer biomarkers discovery.
Authors: Tam Rachel Nga Wan , Schleifman Erica , Huw Ling-Yuh , Desai Rupal , Patel Rajesh , Raja Rajiv
Published: August 2014

Molecular profiling plays an increasingly vital role in determining treatment options in oncology. These include expression profiling, mutation detection as well as copy number variation analysis (CNV). Hence there is an increasing need for a robust and high-throughput technology to detect oncogenic hotspot mutations - and copy number variation (CNV) in various kinds of cancer. Although there are commercial assays or kits available to detect genetic aberrations in a given oncogene, there is a need to simultaneously detect these hotspots in more than a few of these genes using very little DNA. Recent technological advances such as next-generation sequencing (NGS) platforms are the powerful tools to unravel the complex biological and genetic mechanisms in different human diseases. However, there are several challenges facing NGS platforms, such as high cost, and DNA input requirement, and difficult data analysis pipeline which require much longer data turnaround time that is not feasible in the clinical setting.

We have developed a high-throughput microfluidic panel for detecting 120 somatic mutations across 11 genes of therapeutic interest using TaqMan or allele-specific PCR (AS-PCR) technologies. The 11-gene mutation panel is designed to detect hotspot mutations that have higher oncogenic potential. Additionally, we can detect (CNV) changes in a total of 18 genes. This mutation panel only requires as little as 2 ng of high quality (fresh frozen) or 100 ng of formalin-fixed, paraffin-embedded (FFPE) genomic DNA. A pre-amplification of 38 amplicons is carried out to enrich DNA carrying the targeted mutations, using custom-designed primer sets followed by detection of the mutations using TaqMan/AS-PCR assays carried out on a Biomark microfluidics system. Mutation, CNV calls including an automated data analysis process have been implemented to run 88 samples per day. We will present data showing validation of this platform using plasmids and archival samples, including correlation between amplified and unamplified samples on alternate mutation detection platforms that will demonstrate sensitivity and selectivity. The platform is being routinely used in our labs to analyze tissues from patients enrolled in clinical trials.

Citation Format: Rachel Nga Wan Tam, Erica Schleifman, Ling-Yuh Huw, Rupal Desai, Rajesh Patel, Rajiv Raja. Development of MUT-MAP, a high-throughput multi-analyte mutation panel for cancer biomarkers discovery. [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 4134. doi:10.1158/1538-7445.AM2013-4134
Abstract 4138: Fluorescence laparoscopy with an LED light source enables fluorescence-guided resection of pancreatic cancer, labeled with fluorophore-conjugated antibodies, in mouse models.
Authors: Metildi Cristina A. , Kaushal Sharmeela , Luiken George A. , Talamini Mark A. , Hoffman Robert M. , Bouvet Michael
Published: August 2014

Orthotopic mouse models of human pancreatic cancer were established with the BxPC-3 RFP pancreatic cancer cell line. Two weeks after implantation, mice were randomized to undergo bright light laparoscopic resection (BLR) (n=19) or fluorescence-guided laparoscopic resection (FLR) (n=19). Fluorescence laparoscopy was performed with a 495-nm emission filter and a Stryker L9000 LED light source 24 hours after tail vein injection of anti-CEA-Alexa 488 antibodies. Bright light laparoscopy was performed with a Stryker X8000 xenon light source. Pre- and postoperative images were taken with the Olympus OV-100 Small Animal Imaging System to assess completeness of resection. Postoperatively, whole body images were obtained to assess for recurrence and follow tumor progression. Intravital and ex vivo images were obtained at termination to evaluate tumor burden. FLS enabled real-time identification and localization of brightly-fluorescent BxPC-3 RFP tumor labeled with anti-CEA-Alexa 488. Furthermore, there was adequate background illumination for surgical navigation permitting improved resection. At termination at 12 weeks, mice in the FLS group had a smaller pancreatic tumor burden compared to mice from the BLS group (5.75 mm2 vs 28.43 mm2; p=0.012). Mice in the FLS group also experienced decreased local recurrence (47.4% vs 78.9%, p=0.046) and distant recurrence (68.4% vs 94.7%, p=0.045) compared to mice in the BLS group. Fluorescence-guided surgery has significant potential to improve laparoscopic approaches in the treatment of pancreatic cancer.

Citation Format: Cristina A. Metildi, Sharmeela Kaushal, George A. Luiken, Mark A. Talamini, Robert M. Hoffman, Michael Bouvet. Fluorescence laparoscopy with an LED light source enables fluorescence-guided resection of pancreatic cancer, labeled with fluorophore-conjugated antibodies, in mouse models. [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 4138. doi:10.1158/1538-7445.AM2013-4138
Abstract 4139: Could dielectrophoretic testing of oral brush biopsies aid early detection and diagnosis of oral cancer?.
Authors: Graham Karen A. , Fedele Stefano , Porter Stephen R. , Kalavrezos Nicholas , McCaul James A. , Lewis Mark P. , Hughes Michael P. , Labeed Fatima H.
Published: August 2014

Cancer of the oral cavity and pharynx was the 9th most common cancer in US males in 2009, and has a 5-year survival rate of 50%, which has remained relatively unchanged for several decades. This low survival rate is partly attributed to patients being diagnosed in advanced stages of the disease, and has lead to the consensus that early detection and diagnosis are key to improving survival. The objective of this research was to use a technique called dielectrophoresis (DEP) to study the electrophysiological characteristics of cells from normal, dysplastic and cancerous oral mucosa, collected via oral brush biopsies (OBBs) from patients with oral squamous cell carcinoma (OSCC) or oral epithelial dysplasia (OED), and from healthy volunteers. DEP testing involved applying AC electric fields to the cells collected via brush biopsies, whilst they were suspended in a conductivity-controlled medium. Interactions between the cells and the electric field resulted in cellular movement, dependent both on frequency of the applied electric field and cell properties. The electrophysiological properties of the cells’ cytoplasm and membrane were then deduced as a result of analysing this cellular movement. After obtaining favourable ethical opinions from all institutions involved, OBB samples were collected from 61 patients with OSCC or OED and from 12 healthy volunteers. At the time of abstract submission, 38 OSCC OBB and 12 OED OBB samples (grouped as Abnormal samples, N = 50), 36 patient-matched OBB samples of visually healthy oral mucosa from OSCC and OED patients and 21 OBB samples from the mucosa of healthy volunteers (grouped as Normal samples, N = 57) had been successfully tested and studied using DEP. Statistical analysis of these preliminary results of DEP testing showed statistically significant differences (p<0.025) in cell membrane electrophysiological properties of Abnormal versus Normal OBB samples, OSCC samples versus Healthy Normal samples and OED samples versus Healthy Normal samples. These preliminary results suggest that DEP testing of oral brush biopsies has the potential to form the basis of a minimally invasive bench-top diagnostic tool for rapid identification of cancerous and potentially malignant lesions.

Citation Format: Karen A. Graham, Stefano Fedele, Stephen R. Porter, Nicholas Kalavrezos, James A. McCaul, Mark P. Lewis, Michael P. Hughes, Fatima H. Labeed. Could dielectrophoretic testing of oral brush biopsies aid early detection and diagnosis of oral 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 4139. doi:10.1158/1538-7445.AM2013-4139