bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2846: Proliferative recovery after chemotherapy-induced senescence in non-small cell lung cancer (NSCLC) cells as a model of tumor dormancy and disease recurrence
Authors: Saleh Tareq , Thekkudan Theresa , Alotaibi Moureq R. , Gewirtz David A.
Published: July 2016

Lung cancer is the leading cause of cancer-related death in both men and women in the United States. Most lung cancer cases are diagnosed in advanced, inoperable stages and are treated with chemoradiation; while chemoradiation is effective in suppressing tumor progression, recurrence following treatment is not infrequent. As there is no well-accepted preclinical model for tumor dormancy and disease recurrence, we hypothesize that the promotion of transient autophagy and senescence could be developed as a model of tumor growth and recovery following treatment. Studies were performed utilizing H460 NSCLC cells as well as a primary NSCLC cell line isolated and grown in our laboratory from a stage IV adenocarcinoma. After treatment with etoposide (1 uM), growth arrest was accompanied by the robust induction of autophagy and senescence. This growth arrest was transient and was followed by proliferative recovery in the course of several days. Genetic and pharmacological inhibition of autophagy failed to interfere with the ability of the cells to regrow, indicating a non-cytoprotective function of autophagy in response to etoposide (in contrast to the cytoprotective function of autophagy exhibited in response to radiation). Quantification of senescence over time based on C12FDG staining and flow cytometry demonstrated that the reversal of growth arrest coincided with a decline in the extent of senescence. To more precisely define the source of the recovered cells, senescent and non-senescent but growth arrested cells were separated by flow cytometry based on their relative β-galactosidase expression and replated. Both cell populations demonstrated the ability to re-emerge from the growth-arrested state and recover proliferative capacity. These observations suggest that senescence is ultimately a transient process in that at least a subpopulation of tumor cells can and will recover proliferative capacity. Furthermore, the reversibility of therapy-induced senescence (TIS) might prove to be a useful model both in cell culture and in vivo to study tumor dormancy and disease recurrence. Studies are currently in progress to determine the impact of senescence inhibition on tumor recovery as well as sensitivity to subsequent therapy given that recurrent disease tends to be relatively refractory to further treatment.

Citation Format: Tareq Saleh, Theresa Thekkudan, Moureq R. Alotaibi, David A. Gewirtz. Proliferative recovery after chemotherapy-induced senescence in non-small cell lung cancer (NSCLC) cells as a model of tumor dormancy and disease recurrence. [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 2846.
Abstract 2848: Autophagy, senescence and proliferative recovery subsequent to DNA damage in radiation sensitization by PARP inhibition
Authors: Alotaibi Moureq R. , Sharma Khushboo , Saleh Tareq , Povirk Lawrence , Hendrickson Eric , Gewirtz David
Published: July 2016

Radiotherapy continues to be a primary modality in the treatment of cancer. DNA damage induced by radiation can promote apoptosis as well as both autophagy and senescence, where autophagy and senescence can theoretically function to prolong tumor survival. A primary aim of this work was to investigate the hypothesis that autophagy and/or senescence could be permissive for DNA repair, thereby facilitating tumor cell recovery from radiation-induced growth arrest and/or cell death. In addition, studies were designed to elucidate the involvement of autophagy and senescence in radiation sensitization by PARP inhibitors and the re-emergence of a proliferating tumor cell population. In the context of this work, the relationship between radiation-induced autophagy and senescence was also determined. Studies were performed using DNA repair proficient HCT116 colon carcinoma cells and a repair deficient Ligase IV (-/-) isogenic cell line. Irradiation promoted a parallel induction of autophagy and senescence that was strongly correlated with the extent of persistent H2AX phosphorylation in both cell lines; however inhibition of autophagy failed to suppress senescence, indicating that the two responses were dissociable. Irradiation resulted in a transient arrest in the HCT116 cells while arrest was prolonged in the Ligase IV (-/-) cells; however, both cell lines ultimately recovered proliferative function, which may reflect maintenance of DNA repair capacity. The PARP inhibitors (Olaparib) and (Niraparib) increased the extent of persistent DNA damage induced by radiation as well as the extent of both autophagy and senescence; neither cell line underwent significant apoptosis by radiation alone or in the presence of the PARP inhibitors. Inhibition of autophagy failed to attenuate radiation sensitization, indicating that autophagy was not involved in the action of the PARP inhibitors. As with radiation alone, despite sensitization by PARP inhibition, proliferative recovery was evident within a period of 10-20 days. While inhibition of DNA repair via PARP inhibition may initially sensitize tumor cells to radiation via the promotion of senescence, this strategy does not appear to interfere with proliferative recovery, which could ultimately contribute to disease recurrence.

Citation Format: Moureq R. Alotaibi, Khushboo Sharma, Tareq Saleh, Lawrence Povirk, Eric Hendrickson, David Gewirtz. Autophagy, senescence and proliferative recovery subsequent to DNA damage in radiation sensitization by PARP inhibition. [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 2848.
Abstract 2849: RNA-binding protein FXR1 negatively regulates senescence by destabilizing mRNA CDKN1A and stabilizing noncoding RNA telomerase RNA component
Authors: Majumder Mrinmoyee , Palanisamy Nallasivam , Qie Shuo , Day Terry , Diehl Alan J. , Palanisamy Viswanathan
Published: July 2016

RNA-binding proteins (RBPs) regulate numerous aspects of co- and post-transcriptional gene expression. RBP fragile X-related protein 1 (FXR1) belongs to a family of RNA-binding proteins that includes functionally similar Fragile X mental retardation 1 (FMR1) and Fragile X-related 2 (FXR2). FMR1 is significantly studied in Fragile X Syndrome (FXS) where the gene is non-functional due to mutation or aberrant methylation. FXR1 protein is highly expressed in multiple cancers including lung and oral cancers. Here, we demonstrate that RBP FXR1 plays an essential role in the growth of head and neck squamous cell carcinomas (HNSCC) by blocking cellular senescence. FXR1 MEF cells were stained positive for senescence associated beta-galactosidase straining. We report a major function of FXR1 as it promotes the stability of Telomerase RNA Component (TERC), a non-coding RNA and simultaneously destabilizes CDKN1A mRNA, and blocks cellular senescence. FXR1-deficient HNSCC cells show an increase in different cyclin dependent kinase inhibitors (p21, p27), a decrease in p-AKT, and these cells also undergo a G0/G1 cell cycle arrest which are the early onsets of cellular senescence. FXR1 binds and stabilizes TERC RNA for telomere maintenance. On the contrary, FXR1 binds and destabilizes CDKN1A mRNA. By an independent assay we also show that the senescence phenomenon was only observed by a combined up and downregulation of CDKN1A and TERC, respectively which was only obtained by FXR1 knockdown. Thus, FXR1 forms a molecular link between CDKN1A and TERC for cell cycle control and telomere length, respectively, to repress cellular senescence in HNSCC.

Citation Format: Mrinmoyee Majumder, Nallasivam Palanisamy, Shuo Qie, Terry Day, Alan J. Diehl, Viswanathan Palanisamy. RNA-binding protein FXR1 negatively regulates senescence by destabilizing mRNA CDKN1A and stabilizing noncoding RNA telomerase RNA component. [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 2849.
Abstract 2857: The yeast homolog of the mammalian oncogene, Bax Inhibitor-1, regulates the unfolded protein response by altering the ER microenvironment
Authors: Benko Savannah , Brown Melissa , McCarthy Morgan , Sanborn Matthew , Cavedon William , Ritzer Lukas , Berry B. Michael , Austriaco Nicanor
Published: July 2016

Bax inhibitor-1 (BI-1) is an anti-apoptotic gene whose expression is upregulated in a wide range of human cancers. The yeast homolog of BI-1, yeast Bax inhibitor-1 (BXI1), encodes a protein that belongs to the Bax Inhibitor (TMBIM) family of proteins. The crystal structure of a prokaryotic member of the family, BsYetJ, has revealed that the Bax inhibitor proteins are pH sensitive calcium leaks. Our studies have shown that yeast Bxi1p is localized to the ER and is involved in the unfolded protein response (UPR) that is triggered by ER stress. We have recently found that Bxi1p regulates the UPR downstream of endonuclease Ire1p clustering but upstream of HAC1 mRNA splicing. Studies with an ER localized calcium reporter and an ER redox reporter suggest that Bxi1p accomplishes this by altering the ER microenvironment. [Our laboratory is supported by grant NIGMS R15 GM110578, awarded to N. Austriaco.]

Citation Format: Savannah Benko, Melissa Brown, Morgan McCarthy, Matthew Sanborn, William Cavedon, Lukas Ritzer, B. Michael Berry, Nicanor Austriaco. The yeast homolog of the mammalian oncogene, Bax Inhibitor-1, regulates the unfolded protein response by altering the ER microenvironment. [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 2857.
Abstract 2860: Loss of expression of the metastasis suppressor CREB3L1 is associated with high-grade metastatic breast cancer and poorer prognosis
Authors: Mellor Paul , Ward Alison , Anderson Deborah
Published: July 2016

The unfolded protein response (UPR) is required for proper protein folding in the endoplasmic reticulum under conditions of stress such as those encountered in the tumor microenvironment. Recently, a number of studies have suggested a role for the unfolded protein response in the development of cancer, more specifically in regulating the balance between cell death, dormancy and the growth of cancer cells in the tumor microenvironment. This study examined the role of CREB3L1 (cAMP-responsive element-binding protein 3-like protein 1), a member of the UPR, in breast cancer development and metastasis.

Initial experiments identified the loss of CREB3L1 expression in metastatic breast cancer cell lines compared to low- or non-metastatic cell lines. When metastatic cells were transfected with CREB3L1 they demonstrated reduced invasion and migration in vitro, as well as a significantly decreased ability to survive under non-adherent or hypoxic conditions. Interestingly, in an in vivo rat mammary tumor model, CREB3L1 expressing cells not only failed to form metastases compared to CREB3L1 null cells but regression of the primary tumors was seen in 70% of the animals as a result of impaired angiogenesis. Microarray and ChIP on Chip analyses identified changes in the expression of many genes involved in cancer development and metastasis, including a decrease in those involved in angiogenesis.

To determine if these findings translated to human breast cancer, real-time PCR analysis of CREB3L1 expression in human breast cancer tissues of differing grade (n = 213) was performed. Expression of CREB3L1 was elevated in low and medium grade tissue but substantially reduced in high grade tissue, compared to normal. Analysis of the on-line TCGA database of breast cancer samples identified a strong inverse relationship between CREB3L1 gene methylation and the loss of CREB3L1 mRNA expression. Low expression of CREB3L1 was associated with a poorer prognosis with a shorter relapse-free survival for luminal A and triple negative breast cancer patients.

These data suggest that CREB3L1 plays an important role in suppressing tumorgenesis and loss of expression is required for the development of a metastatic phenotype. In addition CREB3L1 expression may be a useful marker for determining patient prognosis.

Citation Format: Paul Mellor, Alison Ward, Deborah Anderson. Loss of expression of the metastasis suppressor CREB3L1 is associated with high-grade metastatic breast cancer and poorer prognosis. [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 2860.
Abstract 2862: The balance of divergent IRE1α signaling regulates pancreatic neuroendocrine tumor growth
Authors: Qi Jenny Y. , Moore Paul C. , Thamsen Maike , Ghosh Rajarshi , Gliedt Micah J. , Maly Dustin J. , Backes Bradley J. , Papa Feroz R. , Oakes Scott A.
Published: July 2016

The Unfolded Protein Response (UPR) is an intracellular signaling pathway that communicates the protein folding status of the endoplasmic reticulum (ER) to the nucleus to maintain ER homeostasis. Hypoxia, nutrient deprivation, proteasome dysfunction, or sustained demands on the secretory pathway–conditions often encountered by solid tumor cells–lead to the accumulation of misfolded proteins in the ER and cause “ER stress.” However, the precise role of UPR signaling in the development and maintenance of cancer remains controversial. We reasoned that pancreatic neuroendocrine tumors (PanNETs) might be one class of solid tumor that may be particularly sensitive to protein folding stress due to their high secretory activity.

The Master UPR regulator IRE1α is an ER transmembrane kinase/endoribonuclease (RNase) that acts as a critical life-death switch depending on the level of ER stress. We analyzed primary human PanNET samples for activation of UPR components and found strong evidence of ER stress and IRE1α hyperactivation in this tumor type. We used a variety of chemical-genetic tools to selectively manipulate IRE1α's homeostatic and apoptotic outputs in a PanNET xenograft model and found that the precise balance of IRE1α signaling is critical for tumor growth in vivo. Disrupting this balance by pushing the pathway in either direction is detrimental to PanNET survival. Moreover, targeting IRE1α with highly selective small molecules in a preclinical mouse model phenocopied the results of genetic manipulation. Our results indicate that IRE1α is an important regulator of PanNET cell survival and growth, and provide a strong rationale for therapeutically targeting this kinase in PanNETs and other cancers that experience high levels of ER stress.

Citation Format: Jenny Y. Qi, Paul C. Moore, Maike Thamsen, Rajarshi Ghosh, Micah J. Gliedt, Dustin J. Maly, Bradley J. Backes, Feroz R. Papa, Scott A. Oakes. The balance of divergent IRE1α signaling regulates pancreatic neuroendocrine tumor growth. [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 2862.
Abstract 2864: Role of ribosomal protein, Rpl22 in regulation of acute lymphoblastic leukemia
Authors: Solanki-Patel Nehal R. , Greenberg Noa , Rao Shu , Peri Suraj , Slifker Michael , Mullighan Charles , Wiest David
Published: July 2016

Inactivation of ribosomal proteins (RP) is known to cause diseases called ribosomopathies, which are often associated with abnormal hematopoiesis and an increased risk for development of leukemia. Our laboratory has recently reported an important link between inactivation of one such ribosomal protein, Rpl22, and poor survival in T cell acute lymphoblastic leukemia (T-ALL) patients. Preliminary bioinformatic analysis shows that RPL22 is lost in ∼10% of pediatric T-ALL patients, who exhibit a more aggressive disease course. Deletion of the RPL22 locus is also enriched in the early T-cell precursor (ETP-ALL) subset of T-ALL patients, which exhibit a more aggressive disease course. Thus, RPL22 loss appears to be a marker for poor outcome in pediatric T-ALL patients. While RP inactivation has previously been linked to increased cancer risk, the mechanistic basis for this linkage remained unclear. We have recently demonstrated that inactivation of Rpl22 promotes leukemic transformation by activating NF-κB and inducing the stem cell gene, LIN28B. Nevertheless, the molecular link between Rpl22 inactivation and the induction of NF-κB activity remained unclear. We have now determined that inactivation of Rpl22 activates NF-κB signaling by exacerbating ER stress responses. Indeed, Rpl22 loss results in hyperactivation of the PERK-EIF2α-ATF4 stress pathway, which is known to activate NF-κB. We have demonstrated that knockdown of PERK using shRNA abrogates the activation of NF-κB in Rpl22 mutant cells and returns Lin28B expression levels to baseline. This reduction in Lin28B eliminates the predisposition to transformation exhibited by Rpl22 mutant cells, as evidenced by decreased formation of colonies in soft agar. Taken together, these data suggest that RPL22 inactivation may serve as a negative prognostic indicator in T-ALL and that pharmacologic targeting of ER stress pathways may represent a novel therapeutic alternative in this molecularly-defined subclass of T-ALL.

Citation Format: Nehal R. Solanki-Patel, Noa Greenberg, Shu Rao, Suraj Peri, Michael Slifker, Charles Mullighan, David Wiest. Role of ribosomal protein, Rpl22 in regulation of acute lymphoblastic 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 2864.
Abstract 2866: Sodium channel γENaC mediates salt induced inflammatory stress in cancer cells
Authors: Amara Suneetha , Tiriveedhi Venkataswarup
Published: July 2016

Chronic inflammation is known to play a critical role in the development of cancer. Recent evidence suggests that high salt in the tissue microenvironment induces chronic inflammatory milieu. Further, high salt intake has been correlated with higher incidence of cardiovascular diseases and cancers. In this report, using breast cancer cell lines, MDA-MB-231 (highly invasive), MCF-7 (poorly invasive), and a normalized breast epithelial cell line, MCF10A, we determined the molecular basis of the potential inflammatory effect of high sodium chloride (NaCl) in the extracellular environment. Dose response studies demonstrated a significantly reduced cell viability (>50%) with external NaCl concentration above 0.25 M. Combined treatment of high NaCl (0.15 M) with sub-effective interleukin (IL)-17 (0.1 nM) induced reactive nitrogen (RNS) and oxygen (ROS) species release along with upregulation of the enzymes iNOS, SOCS and SOD1. Similar effect was not observed with equi-molar mannitol. Western-blot and qRT-PCR analysis demonstrated an elevated expression of an inflammation-specific epithelial sodium channel, γENaC, in MDA-MB-231 (7.1±1.4 fold, p<0.05), MCF-7 (3.9±0.8 fold, p<0.05) and MCF10A (2.2±0.4 fold, p<0.05) cell lines following combined treatment of high NaCl with sub-effective IL-17. This was significantly abrogated by lipid raft depletion and ERK-1/2 knock-down. These culture conditions have also induced cellular expression of cytokines, IL-6 (3 fold), TNFα (6.2 fold); chemokines, CCL5 (2.3 fold), CXCL-12 (2 fold), MIP-1δ (2.7 fold); and angiogenic growth factor, VEGF (2.5 fold). Taken together, these data suggest that high NaCl in the cellular microenvironment induces a γENaC mediated chronic inflammatory response with a potential pro-carcinogenic effect.

Citation Format: Suneetha Amara, Venkataswarup Tiriveedhi. Sodium channel γENaC mediates salt induced inflammatory stress in 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 2866.
Abstract 2869: FLASH-dependent post-translational regulation of the EMT activator ZEB1
Authors: Abshire Camille , Dragoi Ana Maria
Published: July 2016

Understanding the mechanisms of metastasis initiation and tumor persistence despite aggressive treatments is paramount to developing new viable therapeutic targets. Critical to tumor progression is initiation of epithelial-to-mesenchymal transition (EMT), a cellular process characterized by loss of E-cadherin, a critical cell-cell adhesion molecule, gain of migratory properties and chemoresistance. Transcriptional repression of E-cadherin and other genes responsible for maintaining the epithelial phenotype by ZEB1/ZEB2 family is one of the most significant events occurring at the interface between non-metastatic/metastatic cancer and chemo-sensitive/chemo-resistant cancer. Using a novel interdisciplinary high-throughput screen methodology we uncovered novel regulators of E-cadherin. Caspase 8 - associated protein 2 (CASP8AP2 or FLASH) a protein involved in apoptosis and transcriptional control was identified through this screen and we discovered that FLASH depletion resulted in increased E-cadherin levels at the plasma membrane through loss of ZEB1 expression. Subsequent examination of ZEB1 protein expression revealed that ZEB1 is rapidly degraded in FLASH-depleted cells. This process is blocked by proteasome inhibitors suggesting that post-translational modifications (PTMs) and in particular ubiquitination of ZEB1 is responsible for its fast turnover and decreased protein expression in the absence of FLASH. Indeed, ubiquitinated ZEB1 was detected in FLASH-depleted cells in the presence of proteasome inhibitors. Although ZEB1 is degraded through the ubiquitin pathway, its rapid turnover in the absence of FLASH suggests that protective mechanisms are in place to delay and/or prevent this process. Even more, we determined that the atypical ubiquitin E3 ligase complex Skp1-Pam-Fbxo45 is involved in ZEB1 degradation in the absence of FLASH and is upregulated in cells depleted for FLASH. Altogether our data suggests that FLASH can stabilizes ZEB1 indirectly by transcriptional repression of the ubiquitin ligase responsible for its degradation and directly by binding to ZEB1 and masking the recognition site of the ubiquitin ligase. Even more, absence of FLASH reprograms the cells into a more epithelial-like phenotype which is less invasive and is resistant to TGFβ-induced loss of E-cadherin. Not only is FLASH-dependent post-translational regulation of ZEB1 novel, but is also conserved in multiple cells lines from various tissues, making this mechanism an original therapeutic target for cancer metastasis.

Citation Format: Camille Abshire, Ana Maria Dragoi. FLASH-dependent post-translational regulation of the EMT activator ZEB1. [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 2869.
Abstract 2870: Serine/arginine rich proteins show different distribution patterns in stratum of oral epidermoid carcinoma
Authors: Ceja-Lopez Cynthia J. , Lara-Martínez Reyna , Segura-Valdés Lourdes , Ruíz Godoy Rivera Luz , Ambrosio Hernandez Javier A. , Guadarrama-Orozco Jorge Alberto , Ruíz-García Erika , Jiménez García Luis Felipe , Meneses-García Abelardo
Published: July 2016

Serine/arginine proteins increased their activity during the alternative splicing in cell nucleus. The nuclear speckles, also known as interchromatin granule clusters, are enriched with SR splicing factors and are implicated in gene expression. Nuclear speckle formation is developmentally regulated; in certain cases phosphorylated SR proteins are absent from the nucleus and are instead localized at granular structures in the cytoplasm. SR proteins are related with splicing during different types of cancers and metástasis. We search for phosphorylated SR proteins and the nuclear pattern and speckle formation in human oral carcinomas.

Methods: To address nuclear pattern and speckle formation, we performed a series of immunostaining experiments, with SC35 antibody for phosphorylated srsf2 on sections derived from oral epidermoid tumor from 33 patients. The images were obtained with a microscope (Leica TCS SPE/CTR 4000). For imaging cytometry analyses, a Leica LAS AF Lite software (Leica Microsystems) was used to capture images automatically; images and recognition of nuclear speckles were quantitatively analyzed using analysis software (ImageJ). ANOVA analysis was performed for the cellular and staining correlation.

Results: There are significant differences in morphology and distribution of intranuclear mottled pattern of SR proteins in each tissue layer between oral epithelial without neoplasia and oral squamous cell carcinoma (P < 0.0001). We also found that number, size and pattern (p <0.05) is different between the stratum layers of the oral squamous cells, showing more transcriptional activity in the granulosum stratum related with a decreased in nucleus numbers. In oral squamous cells the pattern changes become homogeneous and similar to the basal layer of normal tissue.

We found that is in the granulosum stratum of oral squamous carcinomas where the SR proteins shows more transcriptional activity. This finding could be related with the increased metabolism of this layer.

Citation Format: Cynthia J. Ceja-Lopez, Reyna Lara-Martínez, Lourdes Segura-Valdés, Luz Ruíz Godoy Rivera, Javier A. Ambrosio Hernandez, Jorge Alberto Guadarrama-Orozco, Erika Ruíz-García, Luis Felipe Jiménez García, Abelardo Meneses-García. Serine/arginine rich proteins show different distribution patterns in stratum of oral epidermoid 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 2870.
Abstract 2877: Effects of metabolic stressors on cellular differentiation
Authors: Schvartzman Juan Manuel , Thompson Craig B.
Published: July 2016

Poor differentiation (the divergence of tumor cell morphology from the cellular morphology of the normal tissue of origin) is associated with unfavorable responses to chemotherapy and poor clinical outcomes in solid tumors. Recurrent somatic mutations in the genes encoding for the metabolic enzymes isocitrate dehydrogenase (IDH) 1 and 2 have recently been identified in patients with acute myeloid leukemias, chrondrosarcomas, cholangiocarcinomas and glioblastomas. Point mutations in IDH lead to production of 2-hydroxyglutarate (2HG). 2HG acts as a potent inhibitor of enzymes involved in the removal of chromatin marks associated with transcriptional repression (DNA and Histone H3K9 methylation). Excess 2HG leads to impaired differentiation but very little is known about the underlying mechanisms. Here we use a well established model of cellular differentiation, the differentiation of 10T1/2 fibroblasts into myofibers by the lineage-specifying transcription factor MyoD to study the mechanisms by which IDH mutations impair differentiation. We show that 5-azacytidine induced differentiation of 10T1/2 cells is impaired when mutant IDH2 is expressed and that IDH2 mutations impair the ability of MyoD expression to reprogram 10T1/2 cells into myofibers. We propose that the metabolic landscape of undifferentiated cells determines the ability of the lineage determining factor MyoD to trigger differentiation and that the undifferentiated state of cancer cells is dependent on their unique metabolic profile. Further work aims to a) define the effects of mutant IDH1/2 on the ability of MyoD to program muscle differentiation, b) determine the nature of the tumorigenic effects of IDH1/2 mutations in 10T1/2 cells and c) identify molecular pathways that can overcome differentiation blocks upon activation. Insight from these experiments will outline the mechanism(s) by which metabolic alterations, in the form of 2HG, impair differentiation and drive tumorigenesis. They will also serve as a platform for the identification of metabolic alterations that trigger differentiation. These mechanisms could prove to be novel and readily targetable processes in an array of poorly differentiated and aggressive cancer subtypes where other pharmacological approaches have failed.

Citation Format: Juan Manuel Schvartzman, Craig B. Thompson. Effects of metabolic stressors on cellular differentiation. [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 2877.
Abstract 2879: PIGU modulates radioactive iodine uptake in differentiated thyroid cancers
Authors: AMIT MORAN , Na’ara shorook , Charas Tomer , gil. Ziv
Published: July 2016

Radioactive iodine (RAI) treatment has a significant role in the management of differentiated thyroid carcinoma after surgical treatment. Although there is significant variability between patients, in response to RAI treatment, the mechanism responsible for this phenomenon is unknown. PIGU is an endoplasmic reticulum protein and a component of the GPIT complex, which plays a crucial role in post-translational modification of proteins such as sodium iodide symporter (NIS). Immunohistochemical analysis revealed significantly lower expression of PIGU in papillary carcinomas compared to matched normal thyroid tissue (P<0.001). To study the role of PIGU in RAI uptake of differentiated thyroid cancer, we stably expressed PIGU and mock plasmid in the papillary cancer cell line (K1-PIGU and K1, respectively). Immunofluorescence analysis showed a significant increase in perinuclear expression of PIGU in the K1-PIGU cancer cells compared to controls. Similarly, flow cytometry analysis showed a significant increase in membranous expression of NIS in PIGU transfected cancer cells compared to controls. There were no significant differences in proliferation, invasion or motility between K1-PIGU and K1 cell lines. PIGU expression significantly increased I125 uptake compared to controls both in vitro (1.13 and 1030 ppm, respectively, p< .001) and in vivo (153 and 1.2×105 ppm, respectively, p< .001). Mitogen-activated protein kinase inhibition showed clinically meaningful increases in iodine uptake and retention in a subgroup of patients with thyroid cancer that is refractory to radioiodine. To address the mechanism underlying PIGU down regulation in RAI resistant tumors, we treated K1 and KI-PIGU cells with U0126 MEK inhibitor. This inhibition resulted in a significant increase in PIGU expression and I125 uptake in vitro. Taken together, our results revealed a pivotal role for PIGU in RAI sensitivity of thyroid cancer cells, by modulation of NIS expression.

Citation Format: MORAN AMIT, shorook Na’ara, Tomer Charas, Ziv gil. PIGU modulates radioactive iodine uptake in differentiated thyroid cancers. [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 2879.
Abstract 289: Melphalan and XPO1 inhibition are synergistic in pre-clinical models of multiple myeloma
Authors: Turner Joel G. , Dawson Jana L. , Sullivan Daniel M.
Published: July 2016

Introduction:

Multiple myeloma (MM) accounts for about 10% of all hematologic malignancies, with estimated numbers of new cases and deaths for 2015 in the US at 26,850 and 11,240, respectively. Significant increases in response/survival have been seen over the past several years; however, MM remains incurable and patients ultimately die from progressive disease refractory to anti-myeloma therapy. There is clearly a need for additional effective therapies.

Materials and Methods:

Human MM parental and melphalan (MEL) resistant cell lines were treated with XPO1 inhibitors (XPO1i) KPT330 or KPT8602 +/- MEL and assayed for apoptosis and viability by flow cytometry. Treated cells were assayed for DNA damage by comet assay and phospho-H2AX protein expression. XPO1/MEL treated cells were assayed for p53, NFkB, IKKα, FANCF, and FANCL by Western blot. Cells from patients with newly diagnosed or relapsed/refractory MM treated with XPO1i/MEL were assayed for apoptosis. NOD/SCID-gamma mice with MM tumors were treated with XPO1i/MEL and assayed for tumor growth, survival, and toxicity.

Results:

MM cell viability was decreased synergistically and apoptosis increased by XPO1I/MEL treatment in all MM cell lines tested. XPO1/MEL drug combination significantly increased DNA damage when compared to either MEL or XPO1 alone, as shown by comet assay and increased phospho-H2AX expression. Western blot showed that XPO1i treatment of MM cells increased p53 and decreased NFkB, IKKα, FANCF, and FANCL. MEL-resistant MM cell lines were found to be sensitized by XPO1i to MEL as shown by apoptosis assay (20-fold). CD138+/light chain+ MM cells from newly diagnosed and relapsed/refractory MM patients were also sensitized (5-10 fold) by XPO1i to MEL (apoptosis assay). NOD/SCID-gamma mice challenged with MM tumors demonstrated a strong synergistic antitumor effect in XPO1i/MEL-treated mice, with increased survival and no significant toxicity.

Conclusions:

XPO1i's improved the response of human MM cell lines and patient MM cells to MEL in vitro and ex vivo. It is possible that this synergy may be due to increased nuclear p53 in combination with decreased NFkB and IKKα and decreased DNA repair proteins FANCL and FANCF reversing MEL resistance by the Fanconi Anemia/BRCA pathway. These preliminary data suggest that XPO1i's augment MEL-induced DNA damage and may also block the repair of the DNA damage—both of which could result in synergistic cell kill. Combination therapies using XPO1i, especially the clinical compounds KPT330 (selinexor) and KPT8602 +/- MEL, may significantly improve the treatment outcomes of MM.

Citation Format: Joel G. Turner, Jana L. Dawson, Daniel M. Sullivan. Melphalan and XPO1 inhibition are synergistic in pre-clinical models of multiple myeloma. [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 289.
Abstract 2897: Recurrent transcriptome alterations across multiple cancer types
Authors: Kaczkowski Bogumil , Tanaka Yuji , Kawaji Hideya , Sandelin Albin , Andersson Robin , Itoh Masayoshi , Lassmann Timo , Hayashizaki Yoshihide , Carninci Piero , Forrest Alistair R.R. ,   FANTOM5 Consortium
Published: July 2016

Genes that are frequently deregulated in cancer are clinically attractive as both potential pan-cancer diagnostic markers and therapeutic targets. Here we compared Cap Analysis of Gene Expression (CAGE) profiles from 225 cancer cell lines and 339 corresponding primary cell samples to identify transcripts that are recurrently deregulated in a broad range of cancer types. CAGE is a 5’ sequence tag technology that globally determines transcription start sites (TSS) in the genome and their expression levels. This allowed us to assess novel aspects of the cancer transcriptome.

First, we identified (at promoter resolution) hundreds of protein coding and long non-coding transcripts that are commonly de-regulated in cancer cell lines. Next, we showed that promoters that overlap repetitive elements (especially SINE/Alu and LTR/ERV1 elements) are often upregulated in cancer. In particular, a specific repeat family, REP522 (largely palindromic, unclassified interspersed repeat of ∼1.8Kb in size), was strongly enriched for the most up-regulated promoters. To our knowledge this is the first report implicating REP522 activation in cancer. Then, taking advantage of the fact that CAGE data can be used to estimate the activity of enhancers from balanced bidirectional transcription we identified 90 enhancer RNA producing regions that are recurrently activated in cancer cell lines. With ENCODE ChIA-PET data, we linked 16 of the cancer-activated enhancers to promoters of known cancer related genes.

Finally, to confirm that our results are relevant to clinical tumors we performed complementary analysis in RNA-seq data from 4,055 tumors and 563 normal tissues profiled by The Cancer Genome Atlas (TCGA) and we identified a core set of pan-cancer biomarkers (of both coding and non-coding transcripts) that are recurrently perturbed in both the FANTOM5 and TCGA datasets.

In summary, our extensive transcriptome analysis identified a comprehensive set of candidate biomarkers with pan-cancer potential, and extended the perspective of enhancers and repetitive elements that are recurrently activated during carcinogenesis.

Citation Format: Bogumil Kaczkowski, Yuji Tanaka, Hideya Kawaji, Albin Sandelin, Robin Andersson, Masayoshi Itoh, Timo Lassmann, Yoshihide Hayashizaki, Piero Carninci, Alistair R.R. Forrest, FANTOM5 Consortium. Recurrent transcriptome alterations across multiple cancer types. [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 2897.
Abstract 2899: Selinexor inhibits NF-κB activity by sequestering IkB-a in the nucleus and blocking IkB-a degradation
Authors: Kashyap Trinayan , Argueta Christian , Klebanov Boris , Unger TJ , Link Benjamin , Werman Maxwell , Lee Margaret , Shacham Sharon , Landesman Yosef
Published: July 2016

Background: Selinexor is a small-molecule therapeutic that inhibits XPO1 mediated nuclear export, resulting in nuclear accumulation of tumor suppressor proteins (TSPs) and subsequently cancer cell death while sparing normal cells. It has been previously demonstrated that the inhibitor of NFκB, IκB-, is localized to the cytoplasm by XPO1 in several cancer cell lines and that treatment of cancer cells with selinexor reduces NF-κB transcriptional activity. The mechanism of NF-κB inhibition by selinexor, however, is not fully understood. We hypothesized that nuclear retention of IκB- and down-regulation of IκB- kinase (IKK) in response to selinexor treatment would inhibit NF-κB transcriptional activity.

Methods: U2OS (Osteosarcoma) cells were treated with selinexor in the presence or absence of TNF stimulation and whole protein lysates were analyzed by Western blotting. IκB- localization was evaluated by immunofluorescence microscopy and NF-κB transcriptional activity by ELISA (Thermo Scientific).

Results: TNFα induced the phosphorylation of NF-κB p65 subunit on serine 536 and IκB- on serine 32/36 through IKK. This resulted in the dissociation of IκB-α from NF-κB and led to IκB-α degradation via the 26S proteasome. Free NF-κB could now migrate into the nucleus and initiate transcriptional activation supporting tumorigenicity and inflammation. The IKK kinase is a complex made of two kinases (IKKα and IKKβ) and one regulatory subunit, NEMO/IKKγ. We found that selinexor treatment blocked IKK activity through the down regulation of the IKK (IKKγ) gamma subunit protein levels. This inhibition was dose dependent and prevented IκB- phosphorylation, thereby protecting IκB- from degradation. The protection of intact IκB- from degradation and its forced nuclear accumulation through XPO1 inhibition enabled inhibition of NF-κB transcriptional activity even in the presence of TNFα. Selinexor did not alter the protein levels of IKK or IKK.

Conclusions: Selinexor blocks TNFα induced degradation of IκB- by reducing the levels of IKK. In addition, selinexor increased nuclear IκB- levels through the inhibition of nuclear export. This blocked NF-κB activity and enhanced cancer cell death. We are currently investigating the beneficial effects of combining selinexor with proteasome inhibitors, which are known to prevent IκB- degradation.

Citation Format: Trinayan Kashyap, Christian Argueta, Boris Klebanov, TJ Unger, Benjamin Link, Maxwell Werman, Margaret Lee, Sharon Shacham, Yosef Landesman. Selinexor inhibits NF-κB activity by sequestering IkB-a in the nucleus and blocking IkB-a degradation. [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 2899.
Abstract 2904: The alternative NF-kB2/RELB pathway is activated by LTB/LTB receptor and NIK to promote cell migration and metastasis-related gene expression in HNSCC
Authors: Das Rita , Cheng Tsu-Fan , Coupar Jamie , Saleh Anthony , Clavijo Paul E. , Yang Xinping , Chen Zhong , VanWaes. Carter
Published: July 2016

Head and neck squamous cell carcinomas (HNSCC) are highly inflammatory and preferentially migrate and metastasize to lymph nodes. In this study, we find that LTβ/LTβR/NIK signaling mediates alternative RELB/NF-kB2 activation, which promotes activation of important cancer related genes and migration. We find that LTβ and LTβR are over expressed in subsets of HNSCC tissues and cell lines, and LTβ activates the alternative NF-κB pathway, enhancing nuclear translocation of RELB and NF-kB2/p52. Knockdown of LTβR decreased its target kinase NIK, and downstream NF-kB subunits RELB and NF-kB2/p52 protein expression. Knockdown of NIK protein decreased RELB and p52 protein expression, while LTβ treatment stabilized NIK, RELB and NF-kB2/p52 expression. Consistent with this, knockdown of LTBR and NIK functionally decreased NF-kB reporter gene activity, while treatment of LTB partially restored the NF-kB reporter activity. Notably, knockdown of NIK and RELB by siRNA inhibited cell migration in HNSCC. Since NIK is known as central regulator of the activation of the non-canonical pathway, we tested the effects of a NIK inhibitor 1,3[2H,4H]-Isoquinolinedione on NF-kB function and cell migration. NIK inhibitor decreased NIK protein in the cytoplasm, downstream nuclear expression of RELB and NF-kB2/p52 proteins by Western blot and RELB localization by immunofluorescence staining. We have found evidence for LTβ induction of migration/metastasis and cell death genes MET, BIRC3,and SERPINE1, and NIK knockdown inhibited cell migration and the inducible expression of MET, BIRC3, SERPINE 1 by LTB. Our data reveal the mechanisms how LTβ and NIK activation mediated alternative NF-kB pathway and contribute to migration and metastasis of HNSCC.

Supported by NIDCD intramural Project ZIA-DC-000016, 73 and 74.

Citation Format: Rita Das, Tsu-Fan Cheng, Jamie Coupar, Anthony Saleh, Paul E. Clavijo, Xinping Yang, Zhong Chen, Carter VanWaes. The alternative NF-kB2/RELB pathway is activated by LTB/LTB receptor and NIK to promote cell migration and metastasis-related gene expression in HNSCC. [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 2904.
Abstract 2914: Biphenylene and bipyridine connected benzofuran compounds as novel regulators of kRAS transcription
Authors: Batra Harshul , Islam Mohammad K. , Thurston David E. , Rahman Khondaker M. , Brooks Tracy A.
Published: July 2016

Pancreatic cancer is the fourth most deadly cancer with 5 year survival rate of ∼6%. One of the major attributes of pancreatic cancer is kRAS mutations. kRAS is a proto-oncogene with intrinsic GTPase activity, and is responsible for cell proliferation, division, and apoptosis. kRAS mutations are observed in >95% of pancreatic adenocarcinoma and in 30% all human tumors. When mutated it leads to continuous activity and uncontrolled proliferation which results in increased tumorigenicity and poor prognosis. Downregulating kRAS expression has shown to halt proliferation and leads to cellular death in pancreatic cancer models, but to date no small molecule capable of such transcriptional silencing has been described. A novel series of molecules with either biphenylene or bipyridine spacer connecting the terminal benzofuran ring was synthesized using solution phase chemistry. Tertiary amine side chains were incorporated to the C2-position of the benzofuran ring to improve the DNA binding affinity of these compounds. The synthesized compounds were purified using a “catch and release” method employing the sulfonic acid based resins. The eleven novel compounds were screened in parallel for their ability to stabilize G-quadruplex structures in the kRAS promoter, and to downregulate kRAS promoter activity. Two of the compounds increased the thermal profile of non-canonical DNA formations in the promoter region by >5 °C; however, on the whole these compounds did not seem to function as G-quadruplex-stabilizing agents. Eight compounds significantly decreased luciferase expression under the explicit control of the kRAS promoter by up to 75%, by compound BF 4.3, through an as-yet unknown mechanism of action. The compound series was examined for the inhibition of cellular viability in two mutant kRAS pancreatic cancer cell lines - Panc-1 and MIA PaCa-2; in both cell lines the compounds containing the bipyridine spacer had greater cytotoxic effects with IC50's of ∼30 μM, consistently. qPCR is being used to confirm the decrease in promoter activity in native cellular conditions. These agents are intriguing in both their novel scaffold and their unexpected activity decreasing kRAS expression in a mechanism unrelated to higher order DNA structures. Ultimately, down regulation of kRAS transcription will provide a novel therapeutic approach for pancreatic cancer and improve the prognosis of this highly lethal disease.

Citation Format: Harshul Batra, Mohammad K. Islam, David E. Thurston, Khondaker M. Rahman, Tracy A. Brooks. Biphenylene and bipyridine connected benzofuran compounds as novel regulators of kRAS transcription. [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 2914.