bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5174: Involvement of COX-2 in p53 induction and Jnk activation following DNA damage.
Authors: Kim Joohwee , Lee Hyunju , Park Kawngil , Eling Thomas E. , Shim Minsub
Published: August 2014

Cyclooxygenase (COX) is a rate-limiting enzyme in arachidonic acid cascade and exists as two related but unique isoforms, COX-1 and COX-2. While COX-1 is constitutively expressed in many tissues and is believed to play a role in tissue homeostasis, COX-2 is highly inducible by various stimuli including inflammatory cytokines, growth factors, and DNA damage. In addition, aberrant COX-2 expression is frequently observed in many types of cancer, suggesting the role of COX-2 in tumor development and progression. p53 is a well-known tumor suppressor and plays a key role in the control of normal cell's response to environmental or cellular stresses. Although p53 has been shown to regulate COX-2 expression, the modulation of p53 levels by COX-2 has not been determined. In the present study, we have examined the role of COX-2 as an upstream regulator of p53 levels using COX-2 knockout (KO) mouse embryonic fibroblasts (MEFs). DNA damage-induced increase in p53 protein levels were greatly reduced in COX-2 KO MEFs compared to wild type (WT) or COX-1 KO MEFs. Furthermore, a COX-2 specific inhibitor suppressed doxorubicin-induced p53 accumulation and cell death. However, initial induction of p53 accumulation in response to doxorubicin was not altered by deficiency or inhibition of COX-2, suggesting that COX-2 may be involved in maintenance of p53 stability. Doxorubicin-induced accumulation of p53 appeared to be mediated through the activation of Jun-N-terminal Kinase (Jnk). Activation of Jnk by doxorubicin was decreased in COX-2 KO MEFs or by COX-2 inhibition, suggesting that DNA damage induces activation of Jnk by a mechanism that involves COX-2. Our results suggest the existence of novel cross-talk between COX-2 and p53 and identify COX-2 as a potential target for reducing the side effects from chemotherapy.

Citation Format: Joohwee Kim, Hyunju Lee, Kawngil Park, Thomas E. Eling, Minsub Shim. Involvement of COX-2 in p53 induction and Jnk activation following DNA damage. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5174. doi:10.1158/1538-7445.AM2013-5174
Abstract 5189: The transcriptional regulation of the long non-coding RNA HOTAIR in ovarian cancer.
Authors: Ozes Ali R. , Miller Dave , Guo Cong , Bhattrai Anurag , Liu Yunlong , Nephew Kenneth P.
Published: August 2014

The Polycomb Group (PcG) Repressive Complexes 1 and 2 (PRC1 and PRC2) maintain gene expression patterns by epigenetically modifying chromatin. PRC2 suppresses transcription by catalyzing the trimethylation of histone H3 at lysine 27 (H3K27me3), a hallmark of repressed chromatin. Although overexpression of PRC2 components has been implicated in cancer initiation, progression and metastasis how PRC2 is recruited to its target genes remains poorly understood. Recent work suggests the interaction of PRC2 with the long-non coding (lnc) RNA HOX transcript antisense intergenic RNA (HOTAIR) could serve as a potential guide to silence target genes, but the mechanism has yet to be defined. In this study, we performed whole transcriptome RNA-seq analysis of isogenic cisplatin- sensitive and -resistant A2780 ovarian cancer (OC) cell lines. Of the lncRNA examined, HOTAIR was markedly induced in the cisplatin resistant cells (5-fold upregulation compared to the cisplatin-sensitve A2780 parental line). shRNA depletion of HOTAIR resensitized the drug-resistant cells to cisplatin. Based on analysis of the HOTAIR promoter using luciferase assays, we identified two putative NF-κB binding sites and three E-box binding sites. To further investigate whether these sites contributed to HOTAIR expression, platinum-sensitive cells were treated with different growth factors and cytokines (0 to 48hrs) and analyzed for changes in HOTAIR expression. Treatment with TNF-α, a well-known inflammatory cytokine and established activator of NF-κB, resulted in 16 fold-induction of HOTAIR. In accord with these results, we observed significant enrichment of both NF-κB and cMYC on the HOTAIR promoter using ChIP assay. Pharmacological inhibition of cMYC and NF-κB reduced HOTAIR expression by 2-fold, and siRNA depletion of the NF-κB p65 subunit or cMYC similarly reduced (P<0.01) HOTAIR expression. Based on these results, we hypothesize a positive feedback loop for HOTAIR transcriptional regulation: HOTAIR inhibits Iκ-Bα (an inhibitor of NF-κB) allowing the lncRNA to enter the nucleus and activate not only its own expression but also cMYC expression to further drive HOTAIR expression as well as cMYC-inducible genes. We are currently testing this hypothesis. We believe this novel pathway, whose components have been implicated in numerous other cancers, may be involved in cisplatin-resistant OC.

Citation Format: Ali R. Ozes, Dave Miller, Cong Guo, Anurag Bhattrai, Yunlong Liu, Kenneth P. Nephew. The transcriptional regulation of the long non-coding RNA HOTAIR in ovarian cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5189. doi:10.1158/1538-7445.AM2013-5189
Abstract 5199: The biological importance of LRIG1 in lung cancer.
Authors: Kvarnbrink Samuel , Edlund Karolina , Botling Johan , Micke Patrick , Henriksson Roger , Hedman Håkan , Johansson Mikael
Published: August 2014

Leucine-rich repeats and immunoglobulin-like domains (LRIG)-1 is an endogenous inhibitor of epidermal growth factor receptor (EGFR) signaling. LRIG1 is down-regulated in several forms of human cancer. The LRIG gene family also includes LRIG2 and LRIG3. EGFR signaling is important in non-small cell lung cancer (NSCLC) and EGFR tyrosine kinase inhibitors are today considered standard treatment in patients with advanced disease. Since smoking recently was reported to down-regulate LRIG1 gene expression in lung epithelium and squamous cell carcinoma of the lung, the LRIG family of proteins may be important in lung cancer. The objective of the present study was to investigate if LRIG is a prognostic factor in non-small cell lung cancer (NSCLC) and if LRIG1 have any effects of lung cancer growth in vivo and in vitro.

In the present study we analyzed the LRIG protein expression by immunohistochemistry in a tissue microarray (TMA) from surgically treated patients with NSCLC (n=366). To study the effect of LRIG1 expression on tumor growth, an animal model was used. Transgenic mice with an inducible EGFR L858R mutation were crossbred with LRIG1 knockout mice and tumor growth was assessed with micro-MRI. The impact of ectopic LRIG1 expression on lung cancer cell proliferation was studied in vitro.

In the TMA, patients with high LRIG1 expression had a significantly longer median survival than negative cases (p=0.012). In the animal model, tumor progression was significantly faster in LRIG1 knockout mice 60 days after tumor induction (p=0.025) indicating that LRIG1 is of importance in the early tumor progression. Transient ectopic expression of LRIG1 inhibited lung cancer cell proliferation in vitro.

In summary, LRIG1 was a positive prognostic factor in lung cancer and LRIG1 expression inhibited lung cancer growth in vivo and in vitro. This suggests that LRIG1 is of biological importance in lung cancer and that further studies of LRIG1 as a biomarker and tumor suppressor in lung cancer are warranted.

Citation Format: Samuel Kvarnbrink, Karolina Edlund, Johan Botling, Patrick Micke, Roger Henriksson, Håkan Hedman, Mikael Johansson. The biological importance of LRIG1 in lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5199. doi:10.1158/1538-7445.AM2013-5199
Abstract 5223: Elucidating the complex cross-talk between the MAPK and PIK3 pathways.
Authors: Egbert Megan E. , Wynn Michelle L. , Wu Zhi Fen , Schnell Santiago , Merajver Sofia D.
Published: August 2014

Targeted molecular inhibitors have emerged as a leading anti-cancer strategy; however, despite promising pre-clinical data, many targeted inhibitors induce undesirable off-target effects in the clinic. The large number of off-target effects associated with molecular inhibitors was recently termed the ‘‘whack a mole problem’’ because inhibiting one molecular target often unintentionally activates another molecule. It is increasingly clear that the high incidence of off-target effects associated with targeted inhibitors is related to the complex interactions and emergent behaviors inherent to the highly complex and dysregulated intracellular networks of cancer. Both the mitogen activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3K) pathways are known to be dysregulated in cancer. In previous work, we and others have demonstrated that the MAPK pathway promotes motility, invasion, and angiogenic factors while the PI3K pathway plays an important role in controlling anchorage independent growth. In addition, the PI3K pathway plays an essential role in stimulating glucose metabolism and the Warburg effect. We hypothesize that robust interactions exist between these two pathways that influence efficacy and potentially also acquired resistance to targeted therapies. Using a combination of experimental and theoretical techniques, we developed a predictive network model linking growth factor signaling to the MAPK and PI3K pathways as well as to glucose metabolism. Specifically, we constructed a logic-based network of the cross-talk between MAPK and PI3K signaling that relied on a detailed literature survey to identify known molecular interactions as well as proposed interactions and regulatory feedback connections in the literature. We next performed a set of experiments using a normal-like breast epithelial cell line and a series of pathway specific inhibitors with and without growth factor stimulation to validate our model. Finally, we repeated these experiments using a diverse set of breast cancer cell lines and integrated this data to produce a series of cancer networks representative of different stages of breast cancer progression. Our model was able to recapitulate both our own experimental data and published data in the literature using a smaller subset of regulatory feedback mechanisms than we started with. Together, our results suggest that some proposed interactions and feedback mechanisms attributed to MAPK and PI3K cross-talk in the literature may not be valid.

Citation Format: Megan E. Egbert, Michelle L. Wynn, Zhi Fen Wu, Santiago Schnell, Sofia D. Merajver. Elucidating the complex cross-talk between the MAPK and PIK3 pathways. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5223. doi:10.1158/1538-7445.AM2013-5223
Abstract 523: Chaetoglobosin K inhibits VEGF expression in ovarian cancer cells.
Authors: Chen Yi C. , Luo Haitao , Douglas Zachary , Stewart Shannon , Li Zhaoliang
Published: August 2014

Ovarian cancer is the 5th leading cause of cancer deaths for women in America. In search for an effective reagent for prevention and treatment of ovarian cancers, we looked into a mycotoxin, chaetoglobosin K (ChK), and investigated its potential of angioprevention in two ovarian cancer cell lines. Cell viability and necrosis were checked with MTS- and LDH-based assays, respectively, and expression of several genes was analyzed by ELISA or Western Blotting. We found this mycotoxin dramatically toxic, sharply reducing cell viability at concentrations of 0.6 to 13 microM, and causing necrosis in both OVCAR-3 and A2780/CP70 cells at 2 microM. However, when applied at low concentrations (0.5 -2 microM), ChK effectively reduced expression of VEGF and HIF-1-alpha proteins, and inhibited phosphorylation of Akt signaling molecules. While further study on ChK is underway, we hypothesize a potential mycotoxin for prevention and treatment of ovarian cancers.

Citation Format: Yi C. Chen, Haitao Luo, Zachary Douglas, Shannon Stewart, Zhaoliang Li. Chaetoglobosin K inhibits VEGF expression in ovarian cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 523. doi:10.1158/1538-7445.AM2013-523
Abstract 5234: Network interrogations on SNAIL inhibitor GN-25 induced perturbations in HMLE-SNAIL cell line models.
Authors: Azmi Asfar S. , Bollig-Fisher Aliccia , Bao Bin , Joon-Park Bum , Lee SH , Song Gyu-Yong , Mohammad Ramzi M. , Sarkar Fazlul H.
Published: August 2014

HMLEs (HMLE-SNAIL and Kras-HMLE-SNAIL pairs) serve as excellent model system to interrogate the effect of SNAIL targeted agents that reverse epithelial-to-mesenchymal transition (EMT). We developed a SNAIL-p53 interaction inhibitor (GN-25) that was shown to suppress SNAIL function. In this report, using pathway network analysis, we show that GN-25 induces the reversal of EMT to mesenchymal-to-epithelial transition (MET) in well recognized HMLE-SNAIL and Kras-HMLE-SNAIL models. GN-25 induced MET was found to be concurrent with growth inhibition, suppression of spheroid forming capacity and induction of apoptosis. We performed systems and pathway network analysis on mRNA expression by microarrays from GN-25 treated Kras-HMLE-SNAIL cells that showed an orchestrated global re-organization of EMT network genes. The expression signatures were validated at the protein level using confocal microscopy, western blot analysis and time lapse videography, and we found down-regulation of mesenchymal markers such as TWIST1, TWIST2, ZEB-1 concurrently with up-regulation of epithelial marker E-Cadherin. Additionally, RNAi studies validated SNAIL dependent mechanism of action of the drug. Most importantly, GN-25 deregulated many major transcription factors (TFs) such as inhibition of oncogenic TFs Myc, TBX2, NR3C1 and enhancement in tumor suppressor TFs such as SMAD7, DD1T3, CEBPA, HOXA5, TFEB, IRF1, IRF7 and XBP1, resulting in MET as well as induction of cell death. Our systems and network investigations provide solid pre-clinical supportive evidence for clinical application of GN-25 for the reversal of EMT phenotype to MET in aggressive cancers.

Citation Format: Asfar S. Azmi, Aliccia Bollig-Fisher, Bin Bao, Bum Joon-Park, SH Lee, Gyu-Yong Song, Ramzi M. Mohammad, Fazlul H. Sarkar. Network interrogations on SNAIL inhibitor GN-25 induced perturbations in HMLE-SNAIL cell line models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5234. doi:10.1158/1538-7445.AM2013-5234
Abstract 5243: The novel calcium-dependent direct binding of S100B to p90 ribosomal S6 kinase (RSK) decreases MAP kinase-mediated RSK activation in malignant melanoma.
Authors: Pierce Adam D. , Hartman Kira G. , Weber David
Published: August 2014

S100B is an effective and extensively used prognostic marker for melanoma, with increasing serum S100B being predictive of disease stage, increased recurrence, and low overall patient survival. Establishing the mechanism by which S100B alters cell signaling provides insight into how it may facilitate the progression of melanoma and aid in developing new pharmacological drugs to inhibit cancer advancement. To evaluate specific roles for S100B in malignant melanoma, our S100B knock-down and over-expression studies established a correlation between S100B expression and cell viability. ERK phosphorylation was also found to correlate with S100B levels. We have discovered RSK, a downstream ERK target, as a novel binding partner of S100B. RSK is known to regulate MAPK signaling in melanoma but the mechanism is unclear. We observed an inverse relationship between activated and phosphorylated RSK and S100B levels. We used a Ca2+-binding mutant of S100B (E31A + E72A) to demonstrate that the S100B-RSK interaction was calcium-dependent. Pull-down experiments revealed the C-terminal domain of RSK to be necessary for S100B binding. In vitro, S100B was found to reduce ERK-dependent phosphorylation of RSK at Thr573. Our cellular fractionation and immuno-fluorescence studies showed that S100B protein prevented nuclear localization of phosphorylated RSK. These data are consistent with a mechanism in which elevated S100B binds directly to RSK in a calcium-dependent manner, preventing ERK-mediated phosphorylation of RSK and inhibiting RSK localization to the nucleus. We have identified the S100B-RSK interaction as a novel drug target in melanoma cells. We are currently screening compounds for inhibitors of the S100B-RSK interaction.

Citation Format: Adam D. Pierce, Kira G. Hartman, David Weber. The novel calcium-dependent direct binding of S100B to p90 ribosomal S6 kinase (RSK) decreases MAP kinase-mediated RSK activation in malignant melanoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5243. doi:10.1158/1538-7445.AM2013-5243
Abstract 5250: The role of tumor associated macrophage on drug resistant in non-small cell lung cancer .
Authors: Huang Wen-Chien , Chen Yu-Jen
Published: August 2014

Increased evidence suggests that chronic inflammation is associated with cancer development, metastasis, invasion and drug resistant. The possible mechanisms by which inflammation can contribute to carcinogenesis include lymphocytes infiltration, cytokines and chemokines secretion, tissue remodeling and angiogenesis, etc. The inflammatory mediators facilitate the communication between tumor cells and tumor-associated host stromal tissue, thereby accelerating tumor progression. Tumor-associated macrophages (TAM) are key regulators of the link between inflammation and cancer. Previous studies found macrophage in non-small cell lung cancer (NSCLC) specimens by immunohistochemistry staining, and also discovered interplay of cancer cells and macrophages would induce secretion of certain cytokines and chemokines. However, the mechanism of cytokines and chemokines involved with tumor drug resistant in NSCLC is still obscure. It is also unclear the regulatory mechanisms in change of macrophages from M1 to M2 phase affected by tumor cells.

In this study, we attempt to investigate the roles of TAM-mediated molecules using different characteristics of NSCLC cell lines (H2126, CL1-1, H1437, H23, H838, CL1-5, and H2009) as the study model. We will explore TAM mediators in NSCLC cell lines to detect and compare the phenotypic characteristics; then, we may investigate the transcriptional signaling pathways that are associated with expression of TAM mediators. Finally, we will correlate the expression of TAM mediators/proteins that are associated with TAM-mediated regulatory signaling in tumor drug resistant to the outcome of NSCLC patients. We believe that these proteins may serve as therapeutic targets for NSCLC patients in the future.

In our pilot study, we analyzed the secretion stimulation of cytokines (MCP-1, IL-6, IL-7, IL-8, IL-10, and TNF-a) and MMPs (MMP-8, MMP-9, MMP-10 and TIMP-1) from cancer cells under the effect of macrophages. In the current study, we will deeply concern that the ambivalent interactions between TAM phenotypes and biological behavior of cancer cells, because the paradoxical responses between cancer cells and TAMs in a to and fro way are potentially evident.

Citation Format: Wen-Chien Huang, Yu-Jen Chen. The role of tumor associated macrophage on drug resistant in non-small cell lung cancer . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5250. doi:10.1158/1538-7445.AM2013-5250

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 5252: CC-223, a selective and potent mTOR kinase inhibitor, synergizes with 5-Aza and Erlotinib in eight NSCLC lines in vitro.
Authors: Tran Tam , Hege Kristen , Mortensen Deborah , Raymon Heather , Xu Shuichan
Published: August 2014

CC-223 is a potent and selective mTOR kinase inhibitor currently undergoing Phase 1 clinical evaluation. We evaluated combinatorial effect of CC-223 with 5-Aza and erlotinib at three different ratios of the two combining agents in three different orders of addition in eight NSCLC lines. Combinational indices (CIs) were calculated based upon published method (Chou and Talalay, 1984). CC-223 synergized with azacitidine (CI<0.7) in most of the lines at all ratios of combination when azacitidine was added first to the cells (SeqM2). Synergism (CI<0.7) and moderate synergism (0.7<CI<0.85) was also observed in some cell lines when CC-223 was added first (SeqM1) or simultaneously (Sim) with azacitidine; however, SeqM2 seems to generate better synergistic effect across all lines than SeqM1 and Sim. In SeqM2 addition, no significant difference was observed among different ratios of the two agents. CC-223 synergized with erlotinib (CI<0.7) in most of the cell lines at a ratio of 1:30 (CC-223:erlotinib). Synergism and moderate synergism (0.7<CI<0.85) were also observed at other ratios. Order of addition had no impact on the combinatorial effect for CC-223+erlotinib. Overall, the data demonstrated that CC-223 synergizes with azacitidine and erlotinib in multiple NSCLC cell lines in vitro. Order of addition has an impact for CC-223 combination with 5-Aza but not with Erlotinib. On the other hand, ratio of the two combinatorial agents is important for CC-223+erlotinib combination in vitro.

Citation Format: Tam Tran, Kristen Hege, Deborah Mortensen, Heather Raymon, Shuichan Xu. CC-223, a selective and potent mTOR kinase inhibitor, synergizes with 5-Aza and Erlotinib in eight NSCLC lines in vitro. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5252. doi:10.1158/1538-7445.AM2013-5252
Abstract 5261: PKC alpha is implicated in epithelial to mesenchymal transition and erlotinib resistance in non-small cell lung carcinoma cells.
Authors: Abera Mahlet , Sordella Raffaella , Kazanietz Marcelo
Published: August 2014

Overexpression and mutational activation of the epidermal growth factor receptor (EGFR) play important roles in the pathogenesis of non-small-cell lung cancer (NSCLC). For patients with EGFR activating mutations, EGFR tyrosine kinase inhibitors (TKIs) are given as a primary therapy. However, the majority of these patients eventually develop resistance to TKIs. To address a potential role of PKC isozymes in the resistance to TKIs, we used an isogenic model: the NSCLC H1650 cell line and its erlotinib-resistance derivative H1650-M3. H1650-M3 cells display a mesenchymal-like morphology driven by TGF-β signaling (PNAS 107:15535-15540, 2010). Comparison of the expression of PKC isozymes by Western blot between parental and erlotinib-resistant H1650 cells revealed that the M3 derivative displays remarkably high levels of PKCα and reduced levels of PKCδ. qPCR analysis established that PKCα mRNA levels in H1650-M3 cells are ∼ 40-fold higher than in parental cells. In order to investigate a potential involvement of PKCα in TKI resistance we used RNAi. Upon PKCα depletion H1650-M3 cells display a morphology that is consistent with the epithelial phenotype. Moreover, depletion of PKCα from H1650-M3 cells led to significant changes in the expression of EMT markers. Indeed, mRNA levels of vimentin, Zeb2, SNAIL and TWIST were reduced by more than 50 % in erlotinib-resistant cells when PKCα expression was silenced. Analysis of cell viability in response to erlotinib treatment showed that pretreatment with either the pan-PKC inhibitor GF 109203X or the cPKC inhibitor Gö6976 sensitized H1650-M3 to erlotinib. Furthermore, PKCα RNAi depletion markedly sensitized H1650 cells to the TKI. Therefore, PKCα up-regulation in erlotinib-resistant cells may play an important role in driving the resistance to the drug. Our results also highlight a potential role for PKCα in EMT in lung cancer cells.

Citation Format: Mahlet Abera, Raffaella Sordella, Marcelo Kazanietz. PKC alpha is implicated in epithelial to mesenchymal transition and erlotinib resistance in non-small cell lung carcinoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5261. doi:10.1158/1538-7445.AM2013-5261
Abstract 5264: Negative regulation of AMPK-dependent metabolic-stress pathways by MIF and D-DT in non-small cell lung carcinoma.
Authors: Brock Stephanie , Rendon Beatriz , Yaddanapudi Kavitha , Mitchell Robert
Published: August 2014

AMP-activated protein kinase (AMPK) is a nutrient and metabolic stress sensing enzyme activated by the tumor suppressor kinase, LKB1. Because macrophage migration inhibitory factor (MIF) and its functional homolog, D-dopachrome tautomerase (D-DT), have pro-tumorigenic functions in non-small cell lung carcinoma (NSCLC) but have AMPK activating properties in nonmalignant cell types, we set out to investigate this apparent paradox. Our data now suggest that, in contrast to MIF and D-DT's AMPK activating properties in non-transformed cells, MIF and D-DT cooperatively act to inhibit steady state phosphorylation and activation of AMPK in LKB1 wildtype and LKB1 mutant human NSCLC cell lines. Our data further indicate that MIF and D-DT - acting through their shared cell surface receptor, CD74 - antagonize NSCLC AMPK activation by maintaining glucose uptake, ATP production and redox balance resulting in reduced Ca2+/calmodulin-dependent kinase kinase [[Unsupported Character - Symbol Font β]] (CaMKK[[Unsupported Character - Symbol Font β]])-dependent AMPK activation. Combined, these studies indicate that MIF and D-DT cooperate to inhibit AMPK activation in an LKB1-independent manner. This suggests that MIF and D-DT promote a unique and well conserved adaptation pathway that is usurped by the malignant cells in order to counter metabolic stress-induced, AMPK-dependent, tumor suppression. Importantly, because the LKB1 tumor suppressor locus is mutated in over 30% of NSCLC lesions rendering these tumors insensitive to AMPK-dependent growth suppression, the identification of unique AMPK agonists would be expected to have a tremendous impact on NSCLC disease management for clinicians. Taken together, simultaneous therapeutic targeting of both MIF and D-DT represents an innovative and clinically efficacious approach to re-activating metabolic stress-induced tumor suppression in NSCLC malignant lesions.

Citation Format: Stephanie Brock, Beatriz Rendon, Kavitha Yaddanapudi, Robert Mitchell. Negative regulation of AMPK-dependent metabolic-stress pathways by MIF and D-DT in non-small cell lung carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5264. doi:10.1158/1538-7445.AM2013-5264
Abstract 5278: Circulating microRNAs profile as a novel biomarker for osteosarcoma.
Authors: Allen-Rhoades Wendy , Kurenbekova Lyazat , Hilsenbeck Susan , Man Tsz-Kwong , Donehower Lawrence , Yustein Jason T.
Published: August 2014

Osteosarcoma (OS) is the primary bone tumor in children, with approximately 400 cases each year diagnosed in patients under the age of twenty. Current therapies can achieve long-term relapse free survival rates near 75% for those with localized disease, while survival rates for patients with overt metastatic disease at diagnosis are often less than 20%. Without adjuvant chemotherapy, over 80% of children will develop metastatic or recurrent disease and all patients require long term monitoring. However, there are no reliable, non-invasive biological markers to detect the presence or progression of disease, assess therapy response, or provide upfront risk stratification. The present method for detecting and monitoring disease status in OS is radiographic imaging which is often difficult to interpret. MicroRNAs (miRNAs) are evolutionarily conserved, stable, small non-coding RNA molecules that are key post-transcriptional regulators and are ideal candidates for circulating biomarker development due to their stability in plasma, ease of isolation with high sensitivity, and the unique expressions associated with specific disease states. As such, we have developed a signature profile of circulating miRNAs utilizing a novel genetically engineered mouse model of OS that can discriminate between mice with and without OS. Specifically, we have determined that circulating miR-205 is decreased 3.2-fold in mice with disease compared to disease-free mice, whereas miR-574-3p, miR-214, and miR-335-5p are increased 1.4, 1.9, and 2.9 fold respectively in the diseased mice. Finally, based upon prior studies utilizing other cancer models, we believe that we will be able to directly translate these findings from the mouse model to patients afflicted with osteosarcoma.

Citation Format: Wendy Allen-Rhoades, Lyazat Kurenbekova, Susan Hilsenbeck, Tsz-Kwong Man, Lawrence Donehower, Jason T. Yustein. Circulating microRNAs profile as a novel biomarker for osteosarcoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5278. doi:10.1158/1538-7445.AM2013-5278
Abstract 528: Characterization of Δ7/11, a functional prolactin-binding protein.
Authors: Fleming Jodie M. , Ginsburg Erika , McAndrew Christopher , Heger Christopher , Cheston LaShanda , Rodriguez-Canales Jaime , Vonderhaar Barbara K. , Paul Goldsmith
Published: August 2014

Prolactin is essential for normal mammary gland development and differentiation, and has been shown to promote tumor cell proliferation and chemotherapeutic resistance. Soluble isoforms of the prolactin receptor have been reported to regulate prolactin bioavailability by functioning as “prolactin binding proteins.” Included in this category is Δ7/11, a product of alternate splicing of the prolactin receptor primary transcript. However, the direct interactions of prolactin with Δ7/11, and the resulting effect on cell behavior, have not been investigated. Herein, we demonstrate the ability of Δ7/11 to bind prolactin using a novel proximity ligation assay and traditional immunoprecipitation techniques. Biochemical analyses demonstrated that Δ7/11 was heavily glycosylated, similar to the extracellular domain of the primary prolactin receptor, and that glycosylation regulated the cellular localization and secretion of Δ7/11. Low levels of Δ7/11 were detected in serum samples of healthy volunteers, but were undetectable in human milk samples. Expression of Δ7/11 was also detected in six of the 62 primary breast tumor biopsies analyzed; however, no correlation was found with Δ7/11 expression and tumor histotype or other patient demographics. Functional analysis demonstrated the ability of Δ7/11 to inhibit prolactin-induced cell proliferation as well as alter prolactin-induced rescue of cell cycle arrest/early senescence events in breast epithelial cells. Collectively, these data demonstrate that Δ7/11 is a novel regulatory mechanism of prolactin bioavailability and signaling.

Citation Format: Jodie M. Fleming, Erika Ginsburg, Christopher McAndrew, Christopher Heger, LaShanda Cheston, Jaime Rodriguez-Canales, Barbara K. Vonderhaar, Goldsmith Paul. Characterization of Δ7/11, a functional prolactin-binding protein. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 528. doi:10.1158/1538-7445.AM2013-528
Abstract 5290: MicroRNA-495-3p functions as tumor suppressor by targeting multiple oncogenic epigenetic effector molecules in gastric cancer.
Authors: Kim Min Gyu , Kim Seung Jin , Bae Hyun Jin , Noh Ji Heon , Kim Jeong Kyu , Eun Jung Woo , Jung Kwang Hwa , Chang Young Gyoon , Shen Qingyu , Park Won Sang , Lee Jung Young , Nam Suk Woo
Published: August 2014

Epigenetic effector molecules are involved in many biological processes by regulating post-translational histone modification and DNA methylation. Recent evidence has suggested that several epigenetic effectors are aberrantly over-expressed in gastric cancer (GC). However, underlying regulatory mechanisms of those molecules are not completely understood. In this study, we identified miR-495-3p which regulates multiple epigenetic effectors through a comprehensive miRNA profiling analysis with in silico target prediction in GC. The down-regulation of miR-495-3p was demonstrated in a subset of GC tissue, revealing inverse correlation with expression levels of epigenetic effectors that were predicted to be targeted by miR-495-3p. Ectopic overexpression of miR-495-3p in GC cells suppressed protein expression and 3’-UTR (untranslated region) reporter luciferase activity of multiple epigenetic effectors including histone deacetylase 2 (HDAC2), SET and MYND domain-containing protein 3 (SMYD3), DNA methyltransferase 1 (DNMT1) and metastasis-associated protein1 (MTA1). Moreover, miR-495-3p attenuated cancer cell proliferation accompanied with LC3B-II conversion, PARP cleavage, p16INK4A expression and down-regulation of CDK4/6 and cyclin D1, which were implicated in autophagic cell death, apoptosis and cell cycle regulation, respectively. In addition, ectopically over-expressed miR-495-3p inhibits cell migration via suppressing epithelial-mesenchymal transition (EMT)-related molecules, Snail and SLUG. Taken together, we suggest that a loss or down-regulation of endogenous miR-495-3p triggers aberrant overexpression of multiple oncogenic epigenetic effector molecules, and thereby miR-495-3p functions as a tumor suppressor in gastric tumorigenesis.

Citation Format: Min Gyu Kim, Seung Jin Kim, Hyun Jin Bae, Ji Heon Noh, Jeong Kyu Kim, Jung Woo Eun, Kwang Hwa Jung, Young Gyoon Chang, Qingyu Shen, Won Sang Park, Jung Young Lee, Suk Woo Nam. MicroRNA-495-3p functions as tumor suppressor by targeting multiple oncogenic epigenetic effector molecules in gastric cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5290. doi:10.1158/1538-7445.AM2013-5290

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 5297: The caHUB biospecimen pre-analytical variables program.
Authors: Carithers Latarsha , Vaught Jim
Published: August 2014

The Cancer Human Biobank is an infrastructure that the Biorepositories and Biospecimen Research Branch of the Cancer Diagnosis Program at NCI uses to carry out highly specialized tissue collections to support biospecimen research programs. Specifically, the caHUB Biospecimen Pre-analytical Variables Program collects data on how variables in tissue collection, handling, storing and shipping standard operating procedures affect downstream analysis. Phase one of this program will assess variations in formalin fixation procedures. This will include examining the effects of (i) time delay to the start of tissue fixation after resection and (ii) overall tissue formalin fixation time on the signal of key proteomic markers used for diagnostic treatment that will be assessed by immunohistochemistry. Future phases of this study will address a variety of pre-analytical variables and alternate analysis platforms. The results of this project will be used to determine best practices for molecular preservation and lead to the development of evidence-based protocols for optimal collection, processing, and storage of biospecimens.

Citation Format: Latarsha Carithers, Jim Vaught. The caHUB biospecimen pre-analytical variables program. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5297. doi:10.1158/1538-7445.AM2013-5297
Abstract 530: Inhibition of amphiregulin-mediated EGFR signaling suppresses tumorigenicity in breast cancer cells.
Authors: Kappler Christiana S. , Ethier Stephen P.
Published: August 2014

Approximately half of all basal type breast cancers show evidence of autocrine activation of the epidermal growth factor receptor (EGFR) by amphiregulin (AREG). In addition, AREG expression is associated with aggressive and chemoresistant forms of breast cancer. Studies in our laboratory show that when AREG is the activating ligand, EGFR accumulates at the cell surface, resulting in altered receptor trafficking and downstream signaling. To investigate the effect of AREG-mediated activation of EGFR on the phenotype of AREG-expressing cancers, AREG expression was knocked-down in SUM149 cells, a highly tumorigenic and metastatic cell line isolated from a basal subtype inflammatory breast cancer, which expresses high levels of AREG and overexpresses EGFR. Microarray analysis of AREG knockdown cells revealed a number of relevant pathways affected by AREG-EGFR signaling. These pathways include Notch signaling, MAPK activation, IL-1 signaling and multiple components of the Wnt signaling pathway. In particular, knockdown of AREG or inhibition of EGFR signaling in AREG stimulated cells resulted in the upregulation of DKK-1, an antagonist of canonical Wnt signaling, and the expression of several WNT target genes. Flow cytometric analysis of these cells demonstrated that AREG knockdown resulted in a significant reduction in CD44+ cells while simultaneously increasing the non-stem cell fraction of CD44-/CD24+ cells. Because CD44hi/CD24lo is characteristic of undifferentiated cells that have the potential to initiate tumor formation, the tumorigenicity of these cells was investigated by transplanting parental and AREG knock-down SUM149 cells into the mammary fat pads of NOD-SCID mice. The tumorigenic activity of the AREG knock-down derivatives was significantly reduced compared to the parental SUM149 cells. In particular, the AREG knockdown clone that expressed the lowest level of AREG expression and the highest level of DKK-1 expression failed to form tumors in NOD-SCID mice. To investigate the link between AREG-mediated activation of EGFR, receptor trafficking, and downstream signaling pathways that are responsible for changes in expression of WNT pathway genes, we are investigating differences in EGFR phosphorylation and downstream signaling stimulated by AREG versus EGF. In conjunction with our previous findings that AREG-mediated EGFR signaling results in activation of NF-κβ and the upregulation of IL1α/β, our current results suggest a link between these pathways, WNT signaling, and tumorigenicity in SUM-149 cells.

Citation Format: Christiana S. Kappler, Stephen P. Ethier. Inhibition of amphiregulin-mediated EGFR signaling suppresses tumorigenicity in breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 530. doi:10.1158/1538-7445.AM2013-530
Abstract 5309: Characterization of a novel oncogene SPOCK1 in humanhepatocellular carcinoma.
Authors: Guan Xin-Yuan , Li Yan , Chen Leilei , Yuan Yun-Fei
Published: August 2014

CHD1L is as an

SNF-2-like transcription factor involved in development of hepatocellular carcinoma (HCC) and SPOCK1 is one of the genes upregulated by CHD1L. In this study, we found that overexpression of SPOCK1 could be detected in 92/135 (68%) of HCCs, which was significantly associated with advanced clinical stage (P=0.020) and metastasis (P=0.024). A significantly positive correlation (Pearson correlation coefficient 0.619, P<0.001) between the expressions of CHD1L and SPOCK1 was detected by qRT-PCR in this cohort of HCC specimens. ChIP-PCR assay confirmed that CHD1L protein bound directly to the promoter region (nt-1662 to +34) of SPOCK1 and activated transcription. Levels of SPOCK1 mRNA were increased among tumors that became metastatic, compared with those that did not, and among patients with shorter overall and disease-free survival times. Ectopic expression of SPOCK1 in HCC cells increased proliferation, foci formation, and colony formation in soft agar; these cells also formed larger xenograft tumors, more rapidly, in nude mice than control

HCC cells. Silencing SPOCK1 expression with short hairpin RNA had the opposite effects. We found that SPOCK1 prevents apoptosis of HCC cells by activating Akt, to block release of cytochrome c and activation of caspases -9 and -3; these effects were reversed with an Akt inhibitor. HCC cells that overexpressed SPOCK1 expressed higher levels of MMP9, were more invasive in Matrigel assays, and formed more metastatic nodules in immunodeficient mice than control HCC cells.

In conclusion, CHD1L activates expression of SPOCK1, which activates Akt signaling to block apoptosis and invasion by HCC cells, in culture and in mice. Levels of SPOCK1 increase with progression of human HCC. SPOCK1 might be used as a prognostic factor or therapeutic target.

Citation Format: Xin-Yuan Guan, Yan Li, Leilei Chen, Yun-Fei Yuan. Characterization of a novel oncogene SPOCK1 in humanhepatocellular carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5309. doi:10.1158/1538-7445.AM2013-5309
Abstract 5312: Mutational analysis of the tyrosine kinome in serous and clear cell endometrial cancers.
Authors: Rudd Meghan L. , Mohamed Hassan , Price Jessica C. , Urick Mary Ellen , O'Hara Andrea J. , Gallo Matthieu Le ,   NISC Comparative Sequencing Program, Cruz Pedro , Zhang Suiyuan , Mullikin James , Merino Maria J. , Bell Daphne W.
Published: August 2014

Endometrial cancers (ECs) arise from the inner epithelial lining of the uterus. At diagnosis, the majority of ECs are carcinomas of endometrioid, serous, or clear cell histologies, or an admixture of at least two of these histological subtypes. Serous and clear ECs are rare at presentation but contribute disproportionately to mortality from EC. Thus, there is an acute need for novel therapeutic approaches to treat serous and clear cell ECs. Small molecule inhibitors directed against tyrosine kinases have shown clinical efficacy in other types of cancer in which the target kinase is mutationally activated. Here, we used Sanger sequencing to systematically search for somatic mutations within 527 exons that encode the catalytic domains of 85 tyrosine kinases from 23 serous, 10 clear cell, and 5 mixed histology ECs. For mutated genes, we extended the screen to another 73 ECs (21 serous, 10 clear cell, 42 endometrioid). Genes that were mutated in both the primary and secondary screens were resequenced in a tertiary screen to search for mutations in all coding exons among the entire panel of 111 endometrial tumors. Overall, we uncovered nonsynonymous somatic mutations of ACK1, DDR1, and KDR in 5.4%, 3.6%, and 1% of ECs. Serous tumors had mutated ACK1, DDR1 and KDR in 2.3%, 4.5%, and 2.3% of cases. Clear cell tumors had mutated ACK1 in 5% of cases. Endometrioid tumors had mutated DDR1 in 2.3% of cases. Immunoblotting confirmed that ACK1 and DDR1 are endogenously expressed in endometrial cancer cell-lines. Eighty-seven percent (7 of 8) of ACK1 mutations and 50% (2 of 4) of DDR1 mutations are predicted to impact protein function. To our knowledge this is the first report of ACK1 and DDR1 mutations in endometrial cancer. Our findings warrant future studies to determine whether the ACK1 and DDR1 mutations uncovered here are pathogenic.

Citation Format: Meghan L. Rudd, Hassan Mohamed, Jessica C. Price, Mary Ellen Urick, Andrea J. O'Hara, Matthieu Le Gallo, NISC Comparative Sequencing Program, Pedro Cruz, Suiyuan Zhang, James Mullikin, Maria J. Merino, Daphne W. Bell. Mutational analysis of the tyrosine kinome in serous and clear cell endometrial cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5312. doi:10.1158/1538-7445.AM2013-5312
Abstract 5335: miR-31 contributes to immortalization and epithelial-mesenchymal transition of oral keratinocyte.
Authors: Tu Hsi-Feng , Hung Pei-Shi , Liu Chung-Ji , Lin Shu-Chuin , Chang Kuo-Wei
Published: August 2014

Oral squamous cell carcinoma (OSCC) is a prevalent malignancy worldwide. MicroRNAs (miRNAs) are short, non-coding RNAs that regulate gene expression by RNA interference and crucial for tumorigenesis. Our previous studies identified miR-31 as an oncogenic miRNA of OSCC, and the increased miR-31 may be contributive to oral carcinogenic process in the early stages. This study further specified the higher miR-31 expression in dysplastic potential malignant disorder (PMD) tissues than non-dysplastic PMD tissues in oral cavity. In addition, miR-31 in the patient's saliva was significantly higher than control subjects. To elucidate the contribution of miR-31 in modulating the extended growth of normal oral keratinocyte, exogenous expression of both miR-31 and hTERT genes were introduced to normal oral keratinocyte (NOK) culture, which led to immortalization of two NOK lines, designated M31OK1 and M31OK3. These immortalized lines exhibited cellular phenotypes lying between NOKs and OSCC cell lines and diminished expression of senescence-associated β-galactosidase. Knockdown of hTERT and/or miR-31 decreased the growth and migration of both lines. M31OK1 also carries p53 gene mutation in codon 273, exhibiting the tolerance to serum-induced senescence, anchorage-independent growth and epithelial-mesenchymal transition (EMT) properties relative to NOK. However, the stemness properties were not increased in M31OK1 in relation to NOKs. Knockdown of miR-31 expression decreased the expression of vimentin and ZEB1 and slightly increased E-cadherin expression in M31OK1 cells. This was correlated with the clinical findings that high miR-31 expression was associated with higher expression of VEGF and lower expression of E-cadherin in PMD tissues. This study concluded that miR-31 may be contributive to early oral carcinogenesis by stimulating factors to facilitate cellular immortalization or by modulating the expression of EMT markers in oral keratinocytes.

Citation Format: Hsi-Feng Tu, Pei-Shi Hung, Chung-Ji Liu, Shu-Chuin Lin, Kuo-Wei Chang. miR-31 contributes to immortalization and epithelial-mesenchymal transition of oral keratinocyte. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5335. doi:10.1158/1538-7445.AM2013-5335