bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2763: DNA methylation profiling unveils TGF-β hyperresponse in tumor associated fibroblasts from lung cancer patients
Authors: Vizoso Miguel , Puig Marta , Carmona F. Javier , Maqueda María , Gomez Antonio , Labernardie Anna , Gabasa Marta , Mendizuri Saioa , Ikemori Rafael , Trepat Xavier , Moran Sebastian , Vidal Enrique , Reguart Noemí , Perera Alexandre , Esteller Manel , Alcaraz Jordi
Published: July 2016

There is growing interest in defining the aberrant molecular differences between normal and tumor-associated fibroblasts (TAFs) that support tumor progression. For this purpose, we recently conducted a genome-wide DNA methylation profiling of TAFs and paired control fibroblasts (CFs) from non-small cell lung cancer (NSCLC) patients, and reported a widespread hypomethylation concomitantly with focal gain of DNA methylation; in addition, we found evidence that a fraction of lung TAFs are fibrocytes in origin. Of note, the aberrant epigenome of lung TAFs had a global impact in gene expression and a selective impact on the TGF-β pathway. To get insights on the functional implications of the latter impact, we analyzed the response of lung TAFs to exogenous TGF-β1 in terms of activation and contractility. We found a larger expression of a panel of activation markers including α-SMA and collagen-I in TAFs compared to control fibroblasts. Likewise, TGF-β1 elicited a larger contractility in TAFs than in CFs as assessed by traction force microscopy. These findings reveal that lung TAFs are hyperresponsive to TGF-β1, which may underlie the expansion and/or maintenance of the tumor-promoting desmoplastic stroma in lung cancer.

Citation Format: Miguel Vizoso, Marta Puig, F. Javier Carmona, María Maqueda, Antonio Gomez, Anna Labernardie, Marta Gabasa, Saioa Mendizuri, Rafael Ikemori, Xavier Trepat, Sebastian Moran, Enrique Vidal, Noemí Reguart, Alexandre Perera, Manel Esteller, Jordi Alcaraz. DNA methylation profiling unveils TGF-β hyperresponse in tumor associated fibroblasts from lung cancer patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2763.
Abstract 2776: The relationship between UHRF1 overexpression and LINE-1 hypomethylation in esophageal squamous cell carcinoma
Authors: Nakamura Kenichi , Baba Yoshifumi , Kosumi Keisuke , Harada Kazuto , Shigaki Hironobu , Miyake Keisuke , Kurashige Junji , Ishimoto Takatsugu , Iwatsuki Masaaki , Yoshida Naoya , Baba Hideo
Published: July 2016

Background: Global DNA hypomethylation contributes to oncogenesis through multiple mechanism. The level of long interspersed nucleotide element-1 (LINE-1) methylation is regarded as a surrogate marker of global DNA methylation, and is drawing interest as a good indicator for predicting cancer prognosis. However, the mechanism by which LINE-1 (global DNA) methylation is controlled in cancer cells remains to be fully explored. Ubiquitin-like with PHD and RING finger domain 1 (UHRF1) plays an essential role in DNA methylation. Previous studies have demonstrated that UHRF1 overexpression might be a mechanism of underlying DNA hypomethylation in hepatocellular carcinoma, and contribute to poor prognosis. Nonetheless, the epigenetic role of UHRF1 in esophageal squamous cell carcinoma (ESCC) remains unclear.

Methods: Using 16 frozen tissues (matched pairs of cancer and normal epithelium), and 160 curatively resected esophageal cancer specimens, we analyzed the relationship between UHRF1 expression, LINE-1 methylation levels and patient outcome. We measured UHRF1 expression by real-time PCR and immunohistochemistry, and LINE-1 methylation levels by bisulfite- pyrosequencing technology. In addition, to investigate the relationships between UHRF1 expression and LINE-1 methylation level (i.e., global DNA methylation level) using UHRF1 overexpression vector and siRNA for UHRF1 with ESCC tissues and ESCC cell lines.

Results: In ESCC tissues, UHRF1 expression were significantly associated with LINE-1 methylation levels, and UHRF1 overexpression correlated with poor prognosis in our cohort of 160 ESCC patients. Furthermore, UHRF1 vector-mediated overexpression caused LINE-1 (global DNA) hypomethylation, and conversely, UHRF1 knockdown using siRNA increased DNA methylation in ESCC cell lines.

Conclusion: These results may suggest that UHRF1 is an epigenetic key regulator of DNA methylation, moreover, can be applicable to attractive cancer therapies.

Citation Format: Kenichi Nakamura, Yoshifumi Baba, Keisuke Kosumi, Kazuto Harada, Hironobu Shigaki, Keisuke Miyake, Junji Kurashige, Takatsugu Ishimoto, Masaaki Iwatsuki, Naoya Yoshida, Hideo Baba. The relationship between UHRF1 overexpression and LINE-1 hypomethylation in esophageal squamous cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2776.
Abstract 2782: The effect of LINE-1 methylation level on survival in pancreatic cancer
Authors: Yamamura Kensuke , Baba Yoshifumi , Zhou Lei , Zhang Xiaobo , Kosumi Keisuke , Taki Katunobu , Harada Kazuto , Ishimoto Takatsugu , Imai Katsunori , Hashimoto Daisuke , Chikamoto Akira , Beppu Toru , Xiaodong Tan , Baba Hideo
Published: July 2016

Background: Genome-wide DNA hypomethylation plays an important role in genomic instability and carcinogenesis. DNA methylation in the long interspersed nucleotide element-1 (LINE-1) is a good indicator of the global DNA methylation level. In previous studies, we demonstrated that LINE-1 hypomethylation was strongly associated with a poor prognosis in some types of cancers including esophageal cancer, gastric cancer, and hepatocellular carcinoma, supporting its potential role as a prognostic marker. However, the relationship between LINE-1 hypomethylation and clinical outcome in pancreatic cancer remains unclear.

Methods: LINE-1 methylation level in 126 samples resected pancreatic cancers was measured by pyrosequencing assay, and the prognostic value of LINE-1 methylation level in pancreatic cancer was examined.

Results: Pancreatic cancers showed significantly lower LINE-1 methylation levels compared to matched non tumorous parenchyma (P = 0.039; N = 36). Tumoral LINE-1 methylation range was 41.3-92.8 (N = 126; mean: 77.7; median: 78.5; standard deviation: 5.7). However, LINE-1 hypomethylation was not significantly associated with overall survival, cancer specific survival, disease-free survival [log-rank p = 0.30, p = 0.18 and p = 0.50, respectively].

Conclusion: The present result suggested that LINE-1 hypomethylation in pancreatic cancer is not associated with poor prognosis. In this study, our cohort was small, therefore further accumulation of data may be needed in order to confirm the effect of LINE-1 methylation level on survival in pancreatic cancer.

Citation Format: Kensuke Yamamura, Yoshifumi Baba, Lei Zhou, Xiaobo Zhang, Keisuke Kosumi, Katunobu Taki, Kazuto Harada, Takatsugu Ishimoto, Katsunori Imai, Daisuke Hashimoto, Akira Chikamoto, Toru Beppu, Tan Xiaodong, Hideo Baba. The effect of LINE-1 methylation level on survival in pancreatic cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2782.
Abstract 280: Synergistic in vitro activity of MOR209/ES414 in combination with enzalutamide
Authors: Sewell Toddy , Blake Michelle , Gross Jane A. , Endell Jan , Weirather Johannes , Blankenship John W.
Published: July 2016

Metastatic castration-resistant prostate cancer (mCRPC) presents a daunting therapeutic challenge for patients who eventually progress after androgen ablation therapy. Although newer AR antagonists show a survival benefit, these patients ultimately relapse. Thus, it is critical to identify new therapies that will work in combination with AR antagonists or in AR-antagonist resistant settings. MOR209/ES414 is a bispecific ADAPTIR™ (modular protein technology) molecule currently under investigation in a phase 1 clinical trial in mCRPC, including patients that have progressed on enzalutamide. In pre-clinical studies, MOR209/ES414 has previously been shown to redirect T cell cytotoxicity against cells expressing Prostate Specific Membrane Antigen (PSMA). Here, we aimed to determine whether enzalutamide affects PSMA expression on CRPC cells in vitro, and could alter the effectiveness of MOR209/ES414 at inducing T-cell mediated tumor cell death.

To address these questions, we utilized a CRPC cell line, 22Rv1, which has low expression of PSMA and is resistant to enzalutamide. Treatment of 22Rv1 cells with 10 μM enzalutamide for 3 weeks resulted in increased PSMA cell surface expression as measured by flow cytometry. 22Rv1 cells exposed to enzalutamide were also more sensitive to MOR209/ES414-mediated T-cell lysis, with a 25% increase in cytotoxicity over a 4 hour chromium release assay compared to 22Rv1 cells that were not exposed to enzalutamide. A concomitant decrease in EC50 for MOR209/ES414-mediated lysis from 0.8 pM to 0.5 pM was also observed in 22Rv1 cells exposed to enzalutamide.

MOR209/ES414 and enzalutamide were next tested in combination using a PSMA-positive, enzalutamide-sensitive cell line, LNCaP cultured with primary T cells over several days. Suboptimal doses of MOR209/ES414 and enzalutamide were added to the cultures, and surviving LnCaP cells were quantitated 4 days later. Enzalutamide did not interfere with the ability of MOR209/ES414 to target T cells to LNCaP cells, and Combination Index analysis showed the activity of MOR209/ES414 and enzalutamide to be synergistic in vitro. These studies provide a rationale for further examination of combining MOR209/ES414 with enzalutamide, even in enzalutamide-resistant settings.

Citation Format: Toddy Sewell, Michelle Blake, Jane A. Gross, Jan Endell, Johannes Weirather, John W. Blankenship. Synergistic in vitro activity of MOR209/ES414 in combination with enzalutamide. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 280.
Abstract 2845: PRMT5-PTEN molecular pathway regulates senescence and self-renewal of Glioblastoma stem-like cells
Authors: Banasavadi-Siddegowda Yeshavanth Kumar , Russell Luke , Zhang Jianying , Karkhanis Vrajesh A. , Imitola Jaime , Baiocchi Robert , Kaur Balveen
Published: July 2016

PURPOSE: Glioblastoma (GBM) represents the most common and aggressive histologic subtype among malignant astrocytoma and is associated with poor outcomes because of heterogeneous tumor cell population including mature non-stem like cell and immature stem-like cells within the tumor. Thus, it is critical to find new target-specific therapeutic modalities. Protein arginine methyltransferase enzyme 5 (PRMT5) regulates many cellular processes through its methylation activity and its overexpression in GBM is associated with more aggressive disease. Previously, we have shown that silencing of PRMT5 expression in differentiated GBM cell lines results in apoptosis and reduced tumor growth in mice. Here we report the critical role of PRMT5 in differentiated GBM non-stem-like cells (non-GSC) grown in serum and undifferentiated GBM stem-like cells (GSC) grown as neurospheres in vitro.

METHODS: GSC grown in stem-cell media and non-GSC grown in serum were transfected with specified siRNAs. At appropriate time, these cells were tested for in vitro proliferation, self-renewal capacity, apoptosis, cell cycle progression, mRNA expression and protein expression. Also, using intracranial mouse xenograft, tumor forming ability of PRMT5-intacta and depleted GSC and non-GSC was determined.

RESULTS: Our results uncover a very significant role for PRMT5 in GSC self-renewal capacity and proliferation. PRMT5 knockdown in non-GSC led to apoptosis, knockdown in GSC led to G1 cell cycle arrest through upregulation of p27 and hypophoshorylation of Rb protein, leading to senescence. Comparison of impact of PRMT5 on cellular signaling by Human Phospho-Kinase Array and ChIP-PCR revealed that unlike non-GSC, PRMT5 controls both AKT and ERK activity by direct repression of PTEN in GSC. In vivo transient depletion of PRMT5 decreased intracranial tumor size and growth rate in mice implanted with both primary tumor derived GSC and non-GSC.

CONCLUSION: This is the first study to identify PTEN as a potential downstream target of PRMT5 and vital to support both mature and immature GBM tumor cell populations.

Citation Format: Yeshavanth Kumar Banasavadi-Siddegowda, Luke Russell, Jianying Zhang, Vrajesh A. Karkhanis, Jaime Imitola, Robert Baiocchi, Balveen Kaur. PRMT5-PTEN molecular pathway regulates senescence and self-renewal of Glioblastoma stem-like cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2845.
Abstract 2850: Role of shelterin proteins for telomere shortening in chronic lymphocytic leukemia
Authors: Ishdorj Ganchimeg , Kost Sara EF , Zang Yunli , Gibson Spencer B. , Johnston James B.
Published: July 2016

Telomeres are important in maintaining the integrity of the chromosomes. A series of shelterin proteins (TRF1, TRF2, RAP1, POT1a and b, TIPP1 and TIN2), are involved in the maintenance of telomeres. In chronic lymphocytic leukemia (CLL), the telomeres are shorter than in normal B cells, with very short telomeres being associated with poor survival. In the present study, we demonstrate that the leukemia cell telomere length shortens over time were CLL are followed over year, indicating that telomere shortening in these cells is an ongoing process. When CLL cell metaphases were examined by Q-FISH, telomere length shortening was found to affect all chromosomes equally. To determine whether these telomere changes were related to alterations in the shelterin complex, protein levels of the six shelterins were determined in CLL cells and normal pooled B cells. In general, apart from TIN2, the shelterin protein levels were increased in CLL cells and TRF2 levels were higher in IgVH unmutated cells than in mutated cells. TIN2 protein levels were dramatically reduced in CLL cells as compared to normal B cells and an alternative isoform of TIN2 was observed, both in IgVH mutated and unmuted cases. TIN2 targeted RT-PCR followed by sequencing revealed a deletion of the entire exon 2, totaling 105 bp. This abnormality appeared in monoclonal B-cell lymphocytosis (MBL) indicating that this was an early event. In addition, a subgroup of patients showed an increase in the spliced form of TIN2, when followed over time, suggesting that this might provide the cell with a survival advantage. A protein translated from the spliced variant RNA is expected to be deleted for 35 amino acids at the N-terminus, which is the binding site for TRF2. As TIN2 maintains telomere structure by forming a complex of TRF1 and TRF2 with the telomere, this should not occur with the splice variant. Indicating a pathological role of spliced TIN2 in CLL, TRF2 was localized in both the nuclei and cytoplasm in CLL cells compared to localization only to the nucleus in normal B cells. To confirm the function of the spliced TIN2, we have overexpressed the both full length and spliced clones of TIN2 in HEK293 cells and are presently determining the localization of TRF2 and the ability to form a TRF2/TIN2 complex. Thus, these studies demonstrate a unique splice variant of TIN2 in CLL, which potentially may influence the formation of the shelterin complex and contribute to telomere shortening in this disease.

Citation Format: Ganchimeg Ishdorj, Sara EF Kost, Yunli Zang, Spencer B. Gibson, James B. Johnston. Role of shelterin proteins for telomere shortening in chronic lymphocytic leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2850.
Abstract 2861: Validation of PERK as an oncology target: A role for the unfolded protein response in cancer
Authors: Dellamaggiore Ken , Mitchell Petia , Sun Ji-Rong , Jones Jeffrey , Muchamuel Tony , Hollenback David , Tadesse Seifu , Booker Shon , Hong Fang-Tsao , Smith Adrian , Rose Mark , Beltran Pedro , Lipford James R.
Published: July 2016

The Unfolded Protein Response (UPR) is a cellular stress response to stressors that induce accumulation of unfolded proteins in the endoplasmic reticulum (aka ER stress). The UPR protects cells from ER stress by increasing the capacity of the ER and attenuating bulk translation. Intense or unresolved ER stress induces apoptosis through pro-apoptotic factors like CHoP. The UPR is activated in tumors, especially those of hematological origin. PERK, a UPR sensor-kinase, is highly active in these settings and might be an attractive target in oncology.

We have generated multiple potent, selective PERK inhibitor scaffolds. Low doses of PERK inhibitor (< pPERK IC50) activate the downstream pathway, whereas higher doses return the pathway to baseline, resulting in a bell-shaped activity curve for all pathway readouts. The activation phase results in robust, selective killing of tumor cells in vitro and in vivo, likely through sustained translation inhibition and CHoP induction. However, application in the clinic will be challenging due to irreversible toxicity to pancreatic islets at constant, high doses and difficulty managing human dosing through a bell curve.

Emerging data might provide a solution to these challenges. PERK IP-kinase assays demonstrate that compound binding at any dose activates PERK and this activity is retained after compound removal. Exposure modeling in vitro demonstrates that transient dosing followed by compound removal results in a conventional sigmoidal dose-response curve for viability. Intermittent dosing in vivo results in CHoP induction and tumor growth inhibition even at very high doses of PERK, consistent with PERK activation following compound clearance. These findings suggest that optimized scheduling might drive robust tumor growth inhibition with reduced risk of toxicity and facilitate a standard clinical dose escalation.

Citation Format: Ken Dellamaggiore, Petia Mitchell, Ji-Rong Sun, Jeffrey Jones, Tony Muchamuel, David Hollenback, Seifu Tadesse, Shon Booker, Fang-Tsao Hong, Adrian Smith, Mark Rose, Pedro Beltran, James R. Lipford. Validation of PERK as an oncology target: A role for the unfolded protein response in cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2861.
Abstract 2876: The androgen receptor utilizes protein kinase docking sites to constitutively activate Elk1
Authors: Rosati Rayna , Patki Mugdha , Dakshnamurthy Selvakumar , Chari Venkatesh , McFall Thomas , Ratnam Manohar
Published: July 2016

The ETS domain transcription factor Elk1 tethers the androgen receptor (AR) to chromatin, enabling sustained activation of a set of genes critical for growth in established prostate cancer cell lines. This process does not entail the transient hyper-activation of immediate early genes typical of activation of Elk1 through phosphorylation by ERK1/2. We compared the structural requirements for the association of AR and Elk1 with that for activation of Elk1 by phosphorylation using mammalian one- and two-hybrid assays. The critical polypeptide segments were mapped by systematic deletion mutagenesis. The amino-terminal A/B domain of AR, which lacks the ligand-binding domain (LBD), was adequate for association with Elk1 and to activate Elk1-AR target genes in LNCaP prostate cancer cells. The AR A/B domain also supported hormone-independent growth of LNCaP cells reflecting the ability of overexpressed natural splice variants of AR, which also lack LBD, to support prostate tumor growth. The association of AR with Elk1 was optimal in the absence of ERK1/2 and serum response factor (SRF), which are the known binding partners of Elk1. We identified two sites on Elk1 that are critical for its association with the AR A/B domain as well as the whole AR molecule. One of those sites spans amino acid residues 307 to 330, overlapping the D box, which is one of two ERK docking sites. The other site on Elk1 mapped to amino acid residues 372 to 397, overlapping the FXFP motif, which comprises the downstream ERK docking site. The results suggest that AR utilizes ERK docking sites to associate with Elk1 and is exclusive of the interaction of Elk1 with ERK1/2 or SRF. A nuclear receptor could thus function as a transcription factor co-activator by binding at protein kinase docking sites.

Citation Format: Rayna Rosati, Mugdha Patki, Selvakumar Dakshnamurthy, Venkatesh Chari, Thomas McFall, Manohar Ratnam. The androgen receptor utilizes protein kinase docking sites to constitutively activate Elk1. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2876.
Abstract 2884: Role of Kaiso in intestinal tumorigenesis
Authors: Tripathi Manish K. , Reynolds Albert B.
Published: July 2016

p120-catenin is a master regulator of classical cadherin stability and essential for epithelial homeostasis. p120 physically interacts with transcription factor Kaiso (ZBTB33) suggesting a direct or indirect role in transcription, but its precise function with respect to Kaiso is largely unknown. Kaiso belongs to the ZBTB family of transcription factors, most of which have important roles in development and/or cancer. Interestingly, Kaiso is strongly expressed in the intestinal crypt and abruptly downregulated as the cells move up onto the villus and terminally differentiate. Kaiso is constitutively upregulated in nascent adenomas initiated by loss of the tumor suppressor APC, suggesting a link between canonical Wnt signaling, Kaiso upregulation and intestinal tumorigenesis. Here, using ChipSeq and proteomics we describe several candidate Kaiso interactions with a group of proteins that include the tumor suppressor BRCA1, CHD2 (a chromatin remodifier) and the Ets1 oncoprotein, along with preliminary evidence for functional significance relevant to intestinal tumorigenesis.

Citation Format: Manish K. Tripathi, Albert B. Reynolds. Role of Kaiso in intestinal tumorigenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2884.
Abstract 2887: Autonomous nucleolar mechanism of colon cancer cell survival through induction of perinuclear ribosomal protein translation
Authors: Shaiken Tattym E. , Opekun Antone R.
Published: July 2016

Hypertrophic and irregularly shaped nucleoli are characteristic of gastroitinal cancers with poor prognosis. The role of nucleolar hypertrophy in cancer cell survival has not been studied well. By suppressing several cellular pathways we observed a common cellular response mechanism that involves acceleration of perinuclear ribosomal protein synthesis and multifocal nucleoli hypertrophy. Paradoxically, the induction of perinuclear ribosomal protein biosynthesis was observed by inhibition of protein translation, suppression of protein degradation and mitochondrial activity. Acceleration of perinuclear protein translation in combination of nuclear hypertrophy was detected not only in actively dividing cells but also in quiescent, cell cycle arrested cells, and when cytotoxic effects were manifested. Intensification of perinuclear ribosomal protein translation and nucleolar hypertrophy by Rotenone-mediated damage of mitochondria supported a century long Otto Warburg observation that most cancer cells predominantly produce energy by high rate of glycolysis. Cell fractionation permitted detection of a perinuclear wall that provides a structural basis for the perinuclear protein translation. We found that that rapid- and slow-growing colon cancer cells significantly differ by abundance of ribosomes in the perinuclear region. Therefore, the perinuclear set of protein translation is an important mechanism of cancer cell survival that rescues ribosome biogenesis and leads to the drug resistance through nuclear transformation.

Citation Format: Tattym E. Shaiken, Antone R. Opekun. Autonomous nucleolar mechanism of colon cancer cell survival through induction of perinuclear ribosomal protein translation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2887.
Abstract 2889: Loss of 4E-BP1 function promotes EMT and metastasis via translational activation of snail
Authors: Ye Qing , Cai Weijia , She Qing-Bai
Published: July 2016

The cap-dependent translation is frequently deregulated in a variety of cancers associated with tumor progression. However, the molecular mechanisms underlying the translational activation for cancer metastatic progression remains largely elusive. Here, we demonstrate that activation of cap-dependent translation by silencing the translational repressor 4E-BP1 causes cancer epithelial cells to undergo epithelial-mesenchymal transition (EMT), which is associated with selective upregulation of the EMT inducer snail followed by repression of E-cadherin expression and promotion of cell migration and invasion as well as metastasis. Conversely, inhibition of cap-dependent translation by a dominant active mutant 4E-BP1 effectively downregulates snail expression and inhibits cell migratory/invasive capacities and metastasis. Furthermore, dephosphorylation of 4E-BP1 by mTORC1 inhibition or directly targeting the eIF4F translation initiation also profoundly attenuates snail translation/expression and cell motility, whereas knockdown of 4E-BP1 or overexpression of snail significantly rescues the inhibitory effects. Importantly, 4E-BP1-regulated snail expression is not associated with its changes in the level of transcription or protein stability. Together, these findings reveal a novel role of 4E-BP1 in the regulation of EMT and metastasis through translational control of snail expression and activity, and suggest that targeting cap-dependent translation may provide a promising approach for blocking snail-mediated metastatic potential of cancer.

Citation Format: Qing Ye, Weijia Cai, Qing-Bai She. Loss of 4E-BP1 function promotes EMT and metastasis via translational activation of snail. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2889.
Abstract 2894: Impact of honokiol on sialylation in breast cancer cells
Authors: Thulasiraman Padmamalini , Garriga Galen
Published: July 2016

Metastasis is a process in which malignant cells spread form the tumor of origin and colonize at distant organs. One of the diagnostic markers of metastatic progression is the overexpression of a transmembrane protein called Mucin 1 (MUC1). High levels of MUC1 have been found in tumor tissues, including breast cancer, and have been implicated in reduced survival rate. After translation of the protein, carbohydrate groups are attached to MUC1, a process called glycosylation. The resulting glycocalyx, which serves as a protective layer on epithelial surfaces, is involved in cell-cell interactions, signaling and metastasis. Glycosylation patterns differ on MUC1 in normal breast tissue in comparison with MUC1 in breast tumors. Not only do breast tumors have simpler and fewer carbohydrate chains than MUC1 from normal breast epithelial tissue, they also have increased levels of the carbohydrate derivative, sialic acid. Altered addition of sialic acid (sialylation) has been associated with cancer transformation and metastatic progression of the disease. The enzyme, α-2,3 sialyltransferase (ST3Gal I) which adds the α-2,3-linked sialic acids to MUC1 is increased in breast tumors and has been correlated with poor prognosis. Shortening of the carbohydrates units on MUC1 in breast tumors unmasks peptides that would otherwise be covered by the carbohydrates in normal MUC1, and current studies seek to identify antibodies that would directly target MUC1 on the cancer cells and in turn prevent cancer metastasis. Plant derived natural product honokiol is being studied for its anti-tumor activities. To assess the effect of honokiol on ST3Gal I, we treated breast cancer cell lines, MDA-MB-231 and MCF-7 cells with honokiol and analyzed its effect on ST3Gal I mRNA and protein expression using RT-PCR and Western Blotting, respectively. Honokiol suppressed ST3Gal I at the mRNA and protein level in both cancer cell lines. To investigate specifically whether honokiol reduces free sialic acid, we treated mammary carcinoma cells with honokiol and using the Sialic Acid Kit, we observed reduction in sialic acid in honokiol-treated mammary carcinoma cells. Future immunoprecipitation studies are underway to investigate specifically whether honokiol suppresses sialylation of MUC1. We propose that these studies will lead to the understanding on how sialylation of MUC1 alters the cellular signaling processes involved in the transformation of breast cancer and eventually to the spread of the disease.

Citation Format: Padmamalini Thulasiraman, Galen Garriga. Impact of honokiol on sialylation in breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2894.
Abstract 2923: Preclinical validation of Omomyc cell-penetrating peptides as a viable in vivo anti-Myc therapy
Authors: SOUCEK. LAURA
Published: July 2016

Deregulated Myc is associated with most human cancers suggesting that its inhibition would be a useful therapeutic strategy. Indeed, we have shown that Myc inhibition displays extraordinary therapeutic benefit in various transgenic mouse models of cancer (i.e. skin, lung, pancreatic cancer and glioma), without eliciting resistance to therapy, and causes only mild, well-tolerated and reversible side effects in normal tissues. For these studies we employed a dominant negative inhibitor of Myc, called Omomyc, which is an effective inhibitor of Myc transactivation function both in vitro and in vivo. Omomyc has so far been utilized exclusively as a transgene and served as a successful proof of principle. Here we present our current results based on the direct use of Omomyc itself as a peptide displaying unexpected and efficient cell-penetrating activity (OmomycCPP). When tested in vitro, OmomycCPP causes growth arrest and/or death of several cancer cell lines. In vivo, upon intranasal administration, it rapidly biodistributes to the lung and brain, as well as to other tissues of the animals, and - like its transgenic counterpart before - has a notable therapeutic impact on KRasG12D-driven Non-small Cell Lung Cancer (NSCLSC).

In summary, our results show that OmomycCPP represents a novel and viable pharmacological strategy to inhibit Myc in vivo, a strategy that has the potential to be translated rapidly to the clinic.

Citation Format: LAURA SOUCEK. Preclinical validation of Omomyc cell-penetrating peptides as a viable in vivo anti-Myc therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2923.
Abstract 293: EGFR inhibition generates drug-tolerant persister cells by blocking AKT activity and thus inactivating Ets-1 function
Authors: Phuchareon Janyaporn , Eisele David W. , McCormick Frank , Tetsu Osamu
Published: July 2016

In non-small-cell lung cancer (NSCLC)—the leading cause of cancer death worldwide—about 10-20% harbor mutations in epidermal growth factor receptor (EGFR), a receptor tyrosine kinase (RTK). Although treatment with EGFR tyrosine kinase inhibitors (TKIs) had shown promise, drug resistance has been the most important determinant limiting its success. Recent studies have identified the mechanisms of drug resistance to TKIs, but the origin of acquired resistant cells remains to be elucidated. We consider that they must arise from a small subset of surviving populations after EGFR inhibition. Thus, we have studied the mechanism by which the subset remains viable after EGFR inhibition, despite cell death in the vast majority. Our study demonstrates that EGFR inhibition in lung cancer cells generates a drug-tolerant subpopulation by blocking AKT activity and thus inactivating Ets-1 function. The remaining cells enter a dormant, non-dividing state because of the inhibited transactivation of Ets-1 target genes cyclins D1, D3, and E2. Moreover, Ets-1 inactivation inhibits transcription of dual specificity phosphatase 6 (DUSP6), a negative regulator specific for ERK1/2. As a result, ERK1/2 is activated, which combines with c-Src to renew activation of the Ras/MAPK pathway, causing increased cell survival by accelerating Bim protein turnover. Conversely, inhibition of elevated ERK1/2 by the addition of a MEK inhibitor enhances programmed cell death through rewiring apoptotic signaling. These observations may explain why a small subset of quiescent cells can tolerate TKIs, and might suggest that combined treatment of TKI and MEK inhibitor could overcome drug resistance in the population.

Citation Format: Janyaporn Phuchareon, David W. Eisele, Frank McCormick, Osamu Tetsu. EGFR inhibition generates drug-tolerant persister cells by blocking AKT activity and thus inactivating Ets-1 function. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 293.
Abstract 2941: Increased expression of ABCB1/MDR1 could be associated with alectinib resistance in ALK-rearranged lung cancer cells
Authors: Tsuji Takahiro , Ozasa Hiroaki , Sakamori Yuichi , Nomizo Takashi , Yagi Yoshitaka , Nagai Hiroki , Kim Young Hak , Mishima Michiaki
Published: July 2016

Alectinib, a second-generation anaplastic lymphoma kinase (ALK) inhibitor, has shown promising activity in ALK-rearranged non-small cell lung cancer (NSCLC). The mechanisms of drug resistance to alectinib have been still unclear. To explore whether overexpression of ATP-binding cassette (ABC) transporters might provide a potential mechanism of alectinib resistance in lung cancer, we established alectinib-resistant cell lines from NCI-H2228 and primary ALK-rearranged cells and evaluated the expression of ABC transporters.

We established ALK-rearranged NSCLC cell lines (KTOR 1, 2, and 3) from tumor cells in pleural effusion obtained from 3 patients with alectinib naïve ALK-rearranged NSCLC. In the 2 of 3 patients, tumor cells in pleural effusion were also obtained when disease progression was observed during alectinib treatment (KTOR1-RE and KTOR2-RE). Expression profiling of ABC transporters using quantitative real-time polymerase chain reaction (qRT-PCR) revealed that the expression of ABCB1 was significantly increased in KTOR1-RE and KTOR2-RE compared with the parental cells (4.6 and 10.8 folds), but not other ABC transporters. Then, alectinib-resistant NCI-H2228 cell lines (H2228-AR) were established by exposing the parental cells to stepwise-increasing alectinib up to 5μM for 10 months. We found that the ABCB1/MDR1 expression in H2228-AR cells were significantly increased (2.09-16.6 folds) compared with the parental cells using qRT-PCR and immunoblotting. Then, we examined the alternation of alectinib cytotoxicity by inhibition of ABCB1. Knockdown of ABCB1 expression using small interfering RNA and inhibition of ABCB1 by verapamil enhanced alectinib cytotoxicity in H2228-AR cells. Next, the levels of ABCB1 expression after short-term alectinib exposure were examined in NCI-H2228 and the three alectinib naïve ALK-rearranged NSCLC primary cell lines (KTOR 1, 2, and 3). In all the four ALK-rearranged cell lines, exposure to 100 nM alectinib for 72 hours induced increased expression of ABCB1 compared with control medium.

These data suggested that increased expression of ABCB1/MDR1 might be, in part, responsible acquired resistance and sensitivity to alectinib in ALK-rearranged NSCLC cells.

Citation Format: Takahiro Tsuji, Hiroaki Ozasa, Yuichi Sakamori, Takashi Nomizo, Yoshitaka Yagi, Hiroki Nagai, Young Hak Kim, Michiaki Mishima. Increased expression of ABCB1/MDR1 could be associated with alectinib resistance in ALK-rearranged lung cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2941.
Abstract 2951: A comprehensive analysis of proteins involved in innate and acquired resistance to molecularly-targeted drugs in breast cancer cells
Authors: Montazeri Hamid
Published: July 2016

Cancer is the second leading cause of death among Americans, and despite advances in diagnosis, new technologies for early detection, and development of potent molecularly-targeted drugs, survival rates in advanced disease have not been improving at desired rates. Perseverance of cancer cells seem to rely both on their significant heterogeneity at the onset of treatment, and the inherent plasticity (genetic instability) to adopt to environmental conditions and exogenous agents. Cancer is known as a heterogeneous disease. In fact, extensive genetic diversity has been revealed not only between different types of cancer, but also within a single tumor (as diversity in the expression of protein biomarkers). This intra-tumor heterogeneity could be a major obstacle in cancer treatment due to a wide range of responsivity to any specific anticancer agent. This study aims to analyze the heterogeneity and plasticity among a small panel of breast cancer cell lines as a reaction to exposure to a variety of molecularly-targeted agents, which could lead us to identification of the proteins that play major roles in drug resistance in cancer cells.

After initial evaluation of the LC50, we exposed five different breast cancer cell lines to high doses of erlotinib (selective ErbB1/EGFR-inhibitor), vemurafenib (pan Raf inhibitor), everolimus (selective mTORC1 inhibitor), and ruxolitinib (pan JAK inhibitor), an collected the survivors. Using an in-house designed microarray and real-time PCR analysis, we analyzed the protein expression profile of the survivors compared to the untreated population, which revealed proteins involved in innate resistance and the heterogeneity among the population. In an alternative approach, we exposed the cells to gradually increasing doses of these selected agents, affording the cells the opportunity to adapt to the treatment. The resistant cells were then analyzed in a similar fashion to reveal proteins involved in acquired resistance.

Citation Format: Hamid Montazeri. A comprehensive analysis of proteins involved in innate and acquired resistance to molecularly-targeted drugs in breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2951.
Abstract 2963: VB7-756: a Her2-specific diabody armed with deBouganin, a plant toxin with a distinct MOA
Authors: Chooniedass Shilpa , Dillon Rachelle L. , Premsukh Arjune , Entwistle Joycelyn , Adams Gregory P. , Hudson Peter J. , MacDonald Glen C. , Cizeau Jeannick
Published: July 2016

VB7-756 is Targeted Protein Therapeutic (TPT) comprised of a de-immunized form of bouganin (deBouganin), a potent, plant-derived, type I ribosome-inactivating protein (RIP), genetically linked to the C6.5 anti-Her2 diabody via a furin protease sensitive linker. To engineer the optimal diabody TPT format, several constructs were generated to assess the best diabody-deBouganin orientation. All constructs were expressed as a soluble protein in E. coli supernatant and compared with respect to expression level, stability and potency. The optimal configuration consisted of deBouganin genetically linked to the N-terminus of the VH-VL diabody via a furin protease-sensitive linker and was termed VB7-756. VB7-756 potency was analyzed against a panel of breast cancer cell lines with disparate levels of Her-2 expression and compared to that of Trastuzumab chemically linked to either DM1 (T-DM1) or MMAE (T-MMAE). Overall, VB7-756 was more potent than T-DM1 and T-MMAE with high Her-2 expressing tumor cell lines. In contrast to T-DM1, VB7-756 potency was unaffected by the Her2-Her3 dimerization mediated by heregulin. As opposed to T-DM1 and T-MMAE, which showed only minimal cytoxicity, VB7-756 was highly potent in vitro against tumor cells with cancer stem cell properties. To further differentiate the RIP mechanism of action of deBouganin from tubulin inhibitor reagents, tumor cells that escaped T-DM1 and T-MMAE treatment were incubated in the presence of VB7-756. Results revealed that, VB7-756 was cytotoxic against cancer cells surviving T-DM1 or T-MMAE treatment suggesting that deBouganin can overcome mechanisms of resistance developed by small molecule agents. Moreover, VB7-756 was also cytotoxic against tumor initiating cancer cells evading T-DM1 or T-MMAE toxicity by preventing tumorosphere formation. Overall these results demonstrate that deBouganin's distinct MOA could overcome mechanisms of resistance affecting the efficacy of small molecule drugs.

Citation Format: Shilpa Chooniedass, Rachelle L. Dillon, Arjune Premsukh, Joycelyn Entwistle, Gregory P. Adams, Peter J. Hudson, Glen C. MacDonald, Jeannick Cizeau. VB7-756: a Her2-specific diabody armed with deBouganin, a plant toxin with a distinct MOA. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2963.
Abstract 2985: Molecular mechanisms of dianhydrogalactitol (VAL-083) in cancer treatment
Authors: Zhai Beibei , Steino Anne , Bacha Jeffrey , Brown Dennis , Daugaard Mads
Published: July 2016

Dianhydrogalactitol (VAL-083) is a unique bi-functional alkylating agent causing methylation of N7-guanine and inter-strand DNA crosslinks. VAL-083 is a small water-soluble molecule that readily crosses the blood-brain-barrier. In China, VAL-083 is approved as a chemotherapeutic drug for the treatment of chronic myelogenous leukemia (CML) and lung cancer. In the United States, VAL-083 has been evaluated in more than 40 National Cancer Institute (NCI)-sponsored phase I and phase II clinical trials. Preclinical studies and clinical trial data suggested antineoplastic effects of VAL-083 in a variety of malignancies, including lung cancer, brain tumors, leukemia, cervical cancer, and ovarian cancer. Here we report new insight into VAL-083 mechanism of action by showing that VAL-083 leads to irreversible cell cycle arrest and cell death caused by replication-dependent DNA damage.

In lung (H2122, H1792, H23 and A549) and prostate (PC3 and LNCaP) cancer cell lines, VAL-083 treatment caused irreversible cell cycle arrest in late S and G2 phase as measured by propidium iodide (PI) and immunofluorescent (IF) staining in synchronized cultures. Importantly, VAL-083 was cytotoxic to all cell lines tested (IC50 range 3.06 - 25.7 μM). Western blot and IF analyses of DNA repair markers were employed to investigate the DNA damage response induced by VAL-083 in cancer cells. VAL-083 treatment led to phosphorylation of the proximal DNA double-strand break (DSB) sensor Ataxia Telangiectasia Mutated kinase (ATM), the single-strand DNA-binding Replication Protein A (RPA32), and the histone variant H2A.X (γH2A.X). Importantly, the DNA damage was specific to cells in S phase indicating that VAL-083-induced DNA cross-links translates into more severe DNA lesions during replication. Furthermore, S/G2 phase cell cycle arrest and increased phosphorylation of γH2A.X in cancer cells persisted after pulse-treatment with VAL-083, indicating irreversible DNA lesions.

Taken together, VAL-083 displayed broad anti-neoplastic activity in lung and prostate cancer cells through the induction of replication-dependent DNA damage. Elucidation of the molecular mechanisms underlying VAL-083 cytotoxicity in cancer cells will offer help in identifying and predicting efficacy of combination treatments.

Citation Format: Beibei Zhai, Anne Steino, Jeffrey Bacha, Dennis Brown, Mads Daugaard. Molecular mechanisms of dianhydrogalactitol (VAL-083) in cancer treatment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2985.
Abstract 2988: Integrated functional RNAi screening and structural genomics identify cell cycle and kinetochore components co-modulating NF-κB activity as potential therapeutic targets in head and neck squamous cell carcinoma
Authors: Saleh Anthony D. , Carlson Sophie , Cornelius Shaleeka , Cheng Hui , Martin Scott , Ormanoglu Pinar
Published: July 2016

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, with over 600,000 new cases diagnosed each year, and a five year survival of ∼50%. Recent findings in The Cancer Genome Atlas (TCGA) analysis of 279 HNSCC patient samples revealed that components of the NF-κB/REL signaling pathways are aberrantly expressed in about ∼70% of cases, and implicated in expression of pro-proliferative, inflammatory, angiogenic, and therapeutic resistance genes. Alterations are prevalent in this pathway across patient samples; however, the detailed mechanisms that increase NF-κB signaling in HNSCC are not well understood. Recently, we established an NF-κB β-lactamase reporter UM-SCC-1 cell line to identify key regulators of this NF-κB oncogenic signaling. We performed genome-wide RNAi screening using the NF-κB reporter line, and upon validation, most structural components of the TNF receptor complex and downstream NF-κB pathway genes, such as IKKs, were scored highly in the screening. We integrated the functional genomics data with structural mutation and expression data from TCGA to prioritize genes that are frequently deregulated in HNSCC specimens. WNT9/FZD6, NOTCH2, and TGFβR2 and several receptors were identified to cross-activate NF-κB signaling. In addition, we observed novel genes that co-regulate NF-κB activity, which are involved in the cell cycle, such as CDC2, CDC7, WEE1 and CALM2, as well as components of the kinetochore such as NDC80, PLK1, TTK, and AURKA. Among these targets WEE1 kinase is currently under investigation as a therapeutic target in HNSCC using the WEE1 inhibitor AZD1775. To further investigate potential cross-talk between WEE1 cell cycle regulation and NF-κB activation, we have shown that treatment of HNSCC cells with AZD1775 decreases NF-κB transcriptional activation which corresponds to decrease in TNF-inducible phosphorylation of RELA. These changes also correlate with alteration in cell cycle regulation detected by flow cytometry. Mechanistic studies are currently underway to understand how WEE1 signaling connects to NF-κB activation, and what role inhibition of NF-κB signaling plays in AZD1775 activity as a sensitizer to chemo-or radiation therapy for HNSCC.

Supported by NIDCD intramural projects ZIA-DC-000073,74

Citation Format: Anthony D. Saleh, Sophie Carlson, Shaleeka Cornelius, Hui Cheng, Scott Martin, Pinar Ormanoglu. Integrated functional RNAi screening and structural genomics identify cell cycle and kinetochore components co-modulating NF-κB activity as potential therapeutic targets in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2988.