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Ewing sarcoma is a cancer of bone and soft tissue in children driven by EWS::ETS fusion, most commonly EWS::FLI1. Because current cytotoxic chemotherapies are not improving the survival of those with metastatic or recurrent Ewing sarcoma cases, there is a need for novel and more effective targeted therapies. While EWS::FLI1 is the major driver of Ewing sarcoma, EWS::FLI1 has been difficult to target. A promising alternative approach is to identify and target the molecular vulnerabilities created by EWS::FLI1. Here we report that EWS::FLI1 induces the expression of Slit2, the ligand of Roundabout (Robo) receptors implicated in axon guidance and multiple other developmental processes. EWS::FLI1 binds to the Slit2 gene promoter and stimulates the expression of Slit2. Slit2 inactivates cdc42 and stabilizes the BAF chromatin remodeling complexes, enhancing EWS::FLI1 transcriptional output. Silencing of Slit2 strongly inhibited anchorage-dependent and anchorage-independent growth of Ewing sarcoma cells. Silencing of Slit2 receptors, Robo1 and Robo2, inhibited Ewing sarcoma growth as well. These results uncover a new role for Slit2 signaling in stimulating Ewing sarcoma growth and suggest that this pathway can be targeted therapeutically.

Ewing sarcoma is an aggressive cancer of bone and soft tissue in children. It is characterized by the chromosomal translocation between EWS and an Ets family transcription factor, most commonly FLI1. We recently reported that Ewing sarcoma depends on the autocrine signaling mediated by a cytokine, NELL2. NELL2 signaling stimulates the transcriptional output of EWS-FLI1 through the BAF chromatin remodeling complexes. While studying the impact of NELL2 silencing on Ewing sarcoma, we found that suppression of NELL2 signaling induces the expression of endogenous retroviruses (ERVs) and LINE-1 retrotransposons, an interferon response, and growth arrest. We determined that a histone methyltransferase, EZH2, is the critical downstream target of NELL2 signaling in suppressing ERVs, LINE-1, an interferon response, and growth arrest. We show that EZH2 inhibitors induce ERVs, LINE-1, and an interferon response in a variety of cancer types. These results uncover the role for NELL2–EZH2 signaling in suppressing endogenous virus-like agents and an antiviral response, and suggest the potential utility of EZH2 inhibitors in enhancing anti-tumor immunity.

Retinoblastoma is a cancer of the infant retina primarily driven by the loss of the Rb tumor suppressor gene, which is undruggable. Here, we identified an autocrine signaling, mediated by secreted frizzled-related protein 2 (SFRP2), which suppresses nitric oxide and enables retinoblastoma growth. SFRP2 silencing resulted in rapid production of nitric oxide, leading to p53-dependent growth arrest of retinoblastoma cells. We show that coxsackievirus and adenovirus receptor (CXADR) is the cell surface receptor for SFRP2 in retinoblastoma cells, that CXADR functions as a "dependence receptor," transmitting a growth inhibitory signal in the absence of SFRP2, and that the balance between SFRP2 and CXADR determines nitric oxide production. Accordingly, high SFRP2 RNA expression correlates with high-risk histopathologic features in retinoblastoma. Targeting SFRP2 signaling by SFRP2-binding peptides or by a pharmacological inhibitor rapidly induced nitric oxide and profoundly inhibited retinoblastoma growth in orthotopic xenograft models. These results reveal a cytokine signaling pathway that regulates nitric oxide production and retinoblastoma cell proliferation, and is amenable to therapeutic intervention. Citation Format: Panneerselvam Jayabal, Xiuye Ma, Kathryn M. Bondra, Yidong Chen, Peter J. Houghton, Yuzuru Shiio. Nitric oxide suppression by secreted frizzled-related protein 2 drives retinoblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB267.

SUMMARY We report here that the autocrine signaling mediated by growth and differentiation factor 6 (GDF6), a member of the bone morphogenetic protein (BMP) family of cytokines, maintains Ewing sarcoma growth by preventing Src hyperactivation. Surprisingly, Ewing sarcoma depends on the prodomain, not the BMP domain, of GDF6. We demonstrate that the GDF6 prodomain is a ligand for CD99, a transmembrane protein that has been widely used as a marker of Ewing sarcoma. The binding of the GDF6 prodomain to the CD99 extracellular domain results in recruitment of CSK (C-terminal Src kinase) to the YQKKK motif in the intracellular domain of CD99, inhibiting Src activity. GDF6 silencing causes hyperactivation of Src and p21-dependent growth arrest. We demonstrate that two GDF6 prodomain mutants linked to Klippel-Feil syndrome are hyperactive in CD99-Src signaling. These results reveal a cytokine signaling pathway that regulates the CSK-Src axis and cancer cell proliferation and suggest the gain-of-function activity for disease-causing GDF6 mutants., Graphical Abstract, In Brief Ewing sarcoma is driven by the EWS-ETS fusion oncoprotein, but little is known about the extracellular signaling regulating this cancer. Zhou et al. report that the prodomain of GDF6 is a ligand for CD99, inhibiting Src through CSK and maintaining Ewing sarcoma growth in an autocrine fashion.

LMO1 encodes a protein containing a cysteine-rich LIM domain involved in protein-protein interactions. Recent studies have shown that LMO1 functions as an oncogene in several cancer types, including non-small cell lung cancer (NSCLC). However, the function of LMO1 in other histological subtypes of lung cancer, such as small cell lung cancer (SCLC), was not investigated. In analyzing the expression of LMO1 across a panel of lung cell lines, we found that LMO1 expression levels were significantly and dramatically higher in SCLC cells, an aggressive neuroendocrine subtype of lung cancer, relative to NSCLC and normal lung cells. In NSCLC cells, LMO1 mRNA levels were significantly correlated with expression of neuroendocrine differentiation markers. Our in vitro investigations indicated that LMO1 had the general property of promoting cell proliferation in lung cancer cells representing different histological subtypes, suggesting a general oncogenic function of LMO1 in lung cancer. In investigating the clinical relevance of LMO1 as an oncogene, we found that a high tumor level of the LMO1 mRNA was an independent predictor of poor patient survival. These results suggest that LMO1 acts as an oncogene, with expression correlated with neuroendocrine differentiation of lung cancer, and that it is a determinant of lung cancer aggressiveness and prognosis. By combining gene expression correlations with patient survival and functional in vitro investigations, we further identified TTK as mediating the oncogenic function of LMO1 in lung cancer cells.

SUMMARY BAF chromatin remodeling complexes play important roles in chromatin regulation and cancer. Here, we report that Ewing sarcoma cells are dependent on the autocrine signaling mediated by NELL2, a secreted glycoprotein that has been characterized as an axon guidance molecule. NELL2 uses Robo3 as the receptor to transmit critical growth signaling. NELL2 signaling inhibits cdc42 and upregulates BAF complexes and EWS-FLI1 transcriptional output. We demonstrate that cdc42 is a negative regulator of BAF complexes, inducing actin polymerization and complex disassembly. Furthermore, we identify NELL2highCD133highEWS-FLI1high and NELL2lowCD133lowEWS-FLI1low populations in Ewing sarcoma, which display phenotypes consistent with high and low NELL2 signaling, respectively. We show that NELL2, CD133, and EWS-FLI1 positively regulate each other and upregulate BAF complexes and cell proliferation in Ewing sarcoma. These results reveal a signaling pathway regulating critical chromatin remodeling complexes and cancer cell proliferation., Graphical Abstract, In brief BAF complexes play important roles in chromatin remodeling and cancers, but the signaling pathways regulating these complexes are poorly understood. Jayabal et al. report that NELL2-cdc42 signaling regulates BAF complexes and Ewing sarcoma cell growth.

// Zhenze Zhao 1 , Xiuye Ma 2 , Spencer D. Shelton 1 , Derek C. Sung 3 , Monica Li 4 , Daniel Hernandez 5 , Maggie Zhang 6 , Michael D. Losiewicz 7 , Yidong Chen 8 , Alexander Pertsemlidis 2, 9, 10 , Xiaojie Yu 11 , Yuanhang Liu 11 , Liqin Du 1 1 Department of Chemistry and Biochemistry at Texas State University, San Marcos, Texas, USA 2 Greehey Children’s Cancer Research Institute at UT Health Science Center at San Antonio, San Antonio, Texas, USA 3 Division of Nutritional Sciences at Cornell University, Ithaca, New York, USA 4 University of Texas at Austin, Austin, Texas, USA 5 Department of Biology at Texas State University, San Marcos, Texas, USA 6 Department of Biology, College of Sciences, University of Texas at San Antonio, San Antonio, Texas, USA 7 Department of Chemistry & Biochemistry at St. Mary’s University, San Antonio, Texas, USA 8 Department of Epidemiology and Biostatistics, at UT Health Science Center at San Antonio, San Antonio, Texas, USA 9 Department of Pediatrics, at UT Health Science Center at San Antonio, San Antonio, Texas, USA 10 Cellular and Structural Biology, at UT Health Science Center at San Antonio, San Antonio, Texas, USA 11 Graduate School of Biomedical Sciences at UT Health Science Center at San Antonio, San Antonio, Texas, USA Correspondence to: Liqin Du, email: liqindu6@txstate.edu Keywords: neuroblastoma, microRNA, differentiation, MYCN Received: March 22, 2016 Accepted: September 13, 2016 Published: October 15, 2016 ABSTRACT MYCN amplification is the most common genetic alteration in neuroblastoma and plays a critical role in neuroblastoma tumorigenesis. MYCN regulates neuroblastoma cell differentiation, which is one of the mechanisms underlying its oncogenic function. We recently identified a group of differentiation-inducing microRNAs. Given the demonstrated inter-regulation between MYCN and microRNAs, we speculated that MYCN and the differentiation-inducing microRNAs might form an interaction network to control the differentiation of neuroblastoma cells. In this study, we found that eight of the thirteen differentiation-inducing microRNAs, miR-506-3p, miR-124-3p, miR-449a, miR-34a-5p, miR-449b-5p, miR-103a-3p, miR-2110 and miR-34b-5p, inhibit N-Myc expression by either directly targeting the MYCN 3’UTR or through indirect regulations. Further investigation showed that both MYCN-dependent and MYCN-independent pathways play roles in mediating the differentiation-inducing function of miR-506-3p and miR-449a, two microRNAs that dramatically down-regulate MYCN expression. On the other hand, we found that N-Myc inhibits the expression of multiple differentiation-inducing microRNAs, suggesting that these miRNAs play a role in mediating the function of MYCN. In examining the published dataset collected from clinical neuroblastoma specimens, we found that expressions of two miRNAs, miR-137 and miR-2110, were significantly anti-correlated with MYCN mRNA levels, suggesting their interactions with MYCN play a clinically-relevant role in maintaining the MYCN and miRNA expression levels in neuroblastoma. Our findings altogether suggest that MYCN and differentiation-inducing miRNAs form an interaction network that play an important role in neuroblastoma tumorigenesis through regulating cell differentiation.

miR-195 has recently been reported to function as a tumor suppressor in various cancers, including non-small cell lung cancer (NSCLC). However, the mechanisms by which miR-195 represses the tumorigenesis of NSCLC cells are not fully understood. We performed a high-throughput screen using an miRNA mimic library and confirmed the identification of miR-195 as a tumor suppressor in NSCLC. We demonstrated that overexpression or induced expression of miR-195 in lung tumors slows tumor growth and that repression of miR-195 accelerates tumor growth. In addition, we found that knockout of miR-195 promotes cancer cell growth. We demonstrated that miR-195 targets cyclin D3 to cause cell cycle arrest at the G1 phase and that miR-195 targets survivin to induce apoptosis and senescence in NSCLC cells. Overexpression of cyclin D3 or survivin reverses the effects of miR-195 in NSCLC cells. Through the analysis of data from The Cancer Genome Atlas, we confirmed that the expression of miR-195 is lower in tumors than in adjacent normal tissues and that low expression of miR-195 is associated with poor survival in both lung adenocarcinoma and squamous cell carcinoma patients. Specifically, we found that BIRC5, which codes for survivin, is upregulated in both adenocarcinoma and squamous cell carcinoma tissues and that high expression of BIRC5 is associated with poor survival in adenocarcinoma, but not squamous cell carcinoma. In addition, the ratio of miR-195 level to BIRC5 level is associated with both recurrence-free and overall survival in lung adenocarcinoma. Our results suggest that the miR-195/BIRC5 axis is a potential target for treatment of lung adenocarcinoma specifically, and NSCLC in general.

Microtubule-targeting agents (MTAs) are widely used for the treatment of non-small cell lung cancer (NSCLC). The response rate is only ∼25%, mainly attributable to drug resistance. To identify determinants of resistance in NSCLC, we performed a high-throughput screen using a library of miRNA mimics. Here we report that miR-195 synergizes with MTAs to inhibit the growth of NSCLC cells in vitro, that increased expression of miR-195 sensitizes NSCLC cells to MTAs and that repression of miR-195 confers resistance to MTAs. We show that NSCLC tumors over-expressing miR-195 are more sensitive to MTA treatment and that induced expression of miR-195 in NSCLC tumors potentiates the anti-tumor effect of MTAs. Additionally, we demonstrate that miR-195 targets checkpoint kinase 1 (CHEK1) to regulate the response of NSCLC cells to MTAs, that over-expression of CHEK1 contributes to resistance to MTAs and that knock-down of CHEK1 synergizes with MTAs to repress cell growth. Our results highlight the importance of miR-195 in regulating the response of NSCLC cells to MTAs and underline the potential application of miR-195 as a biomarker for response to MTAs, and as a therapeutic adjuvant to MTA treatment.

microRNAs (miRNAs) are small RNAs endogenously expressed in multiple organisms that regulate gene expression largely by decreasing levels of target messenger RNAs (mRNAs). Over the past few years, numerous studies have demonstrated critical roles for miRNAs in the pathogenesis of many cancers, including lung cancer. Cellular miRNA levels can be easily manipulated, showing the promise of developing miRNA-targeted oligos as next-generation therapeutic agents. In a comprehensive effort to identify novel miRNA-based therapeutic agents for lung cancer treatment, we combined a high-throughput screening platform with a library of chemically synthesized miRNA inhibitors to systematically identify miRNA inhibitors that reduce lung cancer cell survival and those that sensitize cells to paclitaxel. By screening three lung cancer cell lines with different genetic backgrounds, we identified miRNA inhibitors that potentially have a universal cytotoxic effect on lung cancer cells and miRNA inhibitors that sensitize cells to paclitaxel treatment, suggesting the potential of developing these miRNA inhibitors as therapeutic agents for lung cancer. We then focused on characterizing the inhibitors of three miRNAs (miR-133a/b, miR-361-3p, and miR-346) that have the most potent effect on cell survival. We demonstrated that two of the miRNA inhibitors (miR-133a/b and miR-361-3p) decrease cell survival by activating caspase-3/7-dependent apoptotic pathways and inducing cell cycle arrest in S phase. Future studies are certainly needed to define the mechanisms by which the identified miRNA inhibitors regulate cell survival and drug response, and to explore the potential of translating the current findings into clinical applications.

Bone morphogenetic proteins (BMPs) promote osteoblast differentiation and bone formation in vitro and in vivo. BMPs canonically signal through Smad transcription factors, but BMPs may activate signaling pathways traditionally stimulated by growth factor tyrosine kinase receptors. Of these, the mTOR pathway has received considerable attention because BMPs activate P70S6K, a downstream effector of mTOR, suggesting that BMP-induced osteogenesis is mediated by mTOR activation. However, contradictory effects of the mTOR inhibitor rapamycin (RAPA) on bone formation have been reported. Since bone formation is thought to be inversely related to lipid accumulation and mTOR is also important for lipid synthesis, we postulated that BMP-7 may stimulate lipogenic enzyme expression in a RAPA-sensitive mechanism. To test this hypothesis, we determined the effects of RAPA on BMP-7-stimulated expression of osteogenic and lipogenic markers in cultured fetal rat calvarial cells. Our study showed that BMP-7 promoted the expression of osteogenic and lipogenic markers. The effect of BMP-7 on osteogenic markers was greater in magnitude than on lipogenic markers and was temporally more sustained. RAPA inhibited basal and BMP-7-stimulated osteogenic and lipogenic marker expression and bone nodule mineralization. The acetyl CoA carboxylase inhibitor TOFA stimulated the expression of osteoblast differentiation markers, whereas palmitate suppressed their expression. We speculate that the BMP-7-stimulated adipogenesis is part of the normal anabolic response to BMPs, but that inappropriate activation of the lipid biosynthetic pathway by mTOR could have deleterious effects on bone formation and could explain paradoxical effects of RAPA to promote bone formation.

Plants of the Amaryllidaceae family produce a large variety of alkaloids and non-basic secondary metabolites, many of which are investigated for their promising anticancer activities. Of these, crinine-type alkaloids based on the 5,10b-ethanophenanthridine ring system were recently shown to be effective at inhibiting proliferation of cancer cells resistant to various pro-apoptotic stimuli and representing tumors with dismal prognoses refractory to current chemotherapy, such as glioma, melanoma, non-small-cell lung, esophageal, head and neck cancers, among others. Using this discovery as a starting point and taking advantage of a concise biomimetic route to the crinine skeleton, a collection of crinine analogues were synthetically prepared and evaluated against cancer cells. The compounds exhibited single-digit micromolar activities and retained this activity in a variety of drug-resistant cancer cell cultures. This investigation resulted in the discovery of new bicyclic ring systems with significant potential in the development of effective clinical cancer drugs capable of overcoming cancer chemotherapy resistance., SCOPUS: ar.j, info:eu-repo/semantics/published

Introduction: microRNAs (miRNAs) are a family of small noncoding RNAs (18-24 nt) that post-transcriptionally repress gene expression by direct binding to the 3' untranslated regions (UTRs) of their targets. By targeting cancer-related genes, miRNAs have been shown not only to regulate cancer growth/progression, but also to modulate the response of cancer cells to chemotherapy. Such miRNAs are potential candidates for therapeutic intervention. Aiming to identify functional miRNAs in non-small cell lung cancer (NSCLC), we performed a high-throughput screen and found that miR-195 inhibits the growth of NSCLC cells and sensitizes them to microtubule-targeting agents (MTAs), a family of chemotherapeutic drugs widely used for NSCLC treatment. The function and mechanism of miR-195 in NSCLC were demonstrated both in vitro and in vivo. Results: We demonstrated that miR-195 synergizes with both a traditional MTA (paclitaxel) and a recently discovered one (eribulin) to repress the growth of NSCLC cells. Overexpression of miR-195 sensitizes NSCLC cells to paclitaxel and eribulin, while knockout of miR-195 confers resistance to paclitaxel and eribulin. Importantly, lung tumors with miR-195 overexpression are more sensitive to eribulin treatment than control tumors. Induced expression of miR-195 in lung tumors potentiates the efficacy of eribulin to repress tumor growth. Additionally, we showed that miR-195 directly targets CHEK1 to regulate the response of NSCLC cells to paclitaxel and eribulin. The direct and specific binding of miR-195 to the 3'UTR of CHEK1 was confirmed by luciferase reporter assay. Repression of CHEK1 using siRNAs and chemical inhibitor synergizes with paclitaxel and eribulin to repress the growth of NSCLC cells. Overexpression of CHEK1 contributes to the resistance to paclitaxel and eribulin in NSCLC cells. Analysis of TCGA data shows that CHEK1 is significantly upregulated in lung tumors compared to adjacent normal tissues and that its upregulation is associated with worse recurrence-free and overall survival. Conclusions: We report the identification of miR-195 as a sensitizer to microtubule-targeting agents in NSCLC, mediated by its repression of CHEK1. Mouse xenografts with induced or constitutive overexpression of miR-195 show that tumors with high miR-195 expression are more sensitive to drug treatment and that induction of miR-195 potentiates the efficacy of eribulin in repressing tumor growth. These results highlight the possible application of miR-195 expression as a biomarker to predict patient response to MTAs and the potential for delivery of miR-195 mimic as an adjuvant to chemotherapy. Citation Format: Xiaojie Yu, Yiqiang Zhang, Xiuye Ma, Alexander Pertsemlidis. MiR-195 potentiates the efficacy of microtubule-targeting agents in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4402.

microRNA-449a (miR-449a) has been identified to function as a tumor suppressor in several types of cancers. However, the role of miR-449a in neuroblastoma has not been intensively investigated. We recently found that the overexpression of miR-449a significantly induces neuroblastoma cell differentiation, suggesting its potential tumor suppressor function in neuroblastoma. In this study, we further investigated the mechanisms underlying the tumor suppressive function of miR-449a in neuroblastoma. We observed that miR-449a inhibits neuroblastoma cell survival and growth through 2 mechanisms--inducing cell differentiation and cell cycle arrest. Our comprehensive investigations on the dissection of the target genes of miR-449a revealed that 3 novel targets- MFAP4, PKP4 and TSEN15 -play important roles in mediating its differentiation-inducing function. In addition, we further found that its function in inducing cell cycle arrest involves down-regulating its direct targets CDK6 and LEF1. To determine the clinical significance of the miR-449a-mediated tumor suppressive mechanism, we examined the correlation between the expression of these 5 target genes in neuroblastoma tumor specimens and the survival of neuroblastoma patients. Remarkably, we noted that high tumor expression levels of all the 3 miR-449a target genes involved in regulating cell differentiation, but not the target genes involved in regulating cell cycle, are significantly correlated with poor survival of neuroblastoma patients. These results suggest the critical role of the differentiation-inducing function of miR-449a in determining neuroblastoma progression. Overall, our study provides the first comprehensive characterization of the tumor-suppressive function of miR-449a in neuroblastoma, and reveals the potential clinical significance of the miR-449a-mediated tumor suppressive pathway in neuroblastoma prognosis.

Fluctuations of intracellular Ca2+ ([Ca2+]i) regulate a variety of cellular functions. The classical Ca2+ transport pathways in the endoplasmic reticulum (ER) and plasma membrane are essential to [Ca2+]i oscillations. Although mitochondria have recently been shown to absorb and release Ca2+ during G protein-coupled receptor (GPCR) activation, the role of mitochondria in [Ca2+]i oscillations remains to be elucidated. Using fluo-3-loaded human teratocarcinoma NT2 cells, we investigated the regulation of [Ca2+]i oscillations by mitochondria. Both the muscarinic GPCR agonist carbachol and the ER Ca2+-adenosine triphosphate inhibitor thapsigargin (Tg) induced [Ca2+]i oscillations in NT2 cells. The [Ca2+]i oscillations induced by carbachol were unsynchronized among individual NT2 cells; in contrast, Tg-induced oscillations were synchronized. Inhibition of mitochondrial functions with either mitochondrial blockers or depletion of mitochondrial DNA eliminated carbachol--but not Tg-induced [Ca2+]i oscillations. Furthermore, carbachol-induced [Ca2+]i oscillations were partially restored to mitochondrial DNA-depleted NT2 cells by introduction of exogenous mitochondria. Treatment of NT2 cells with gap junction blockers prevented Tg-induced but not carbachol-induced [Ca2+]i oscillations. These data suggest that the distinct patterns of [Ca2+]i oscillations induced by GPCR and Tg are differentially modulated by mitochondria and gap junctions.

Epidermal growth factor (EGF) is a multifunctional factor known to influence proliferation and function of a variety of cells. The actions of EGF are mediated by EGF receptor tyrosine kinase pathways, including stimulation of phospholipase Cgamma and mobilization of intracellular Ca(2+) ([Ca(2+)](i)). Generally, agonist-mediated Ca(2+) mobilization involves both Ca(2+) release from internal stores and Ca(2+) influx activated by store depletion (i.e. capacitative or store-operated Ca(2+) influx). However, the role of capacitative Ca(2+) entry in EGF-mediated Ca(2+) mobilization is still largely unknown. In this study, we compared [Ca(2+)](i) signals elicited by EGF with those induced by agents (the muscarinic receptor agonist carbachol and thapsigargin (Tg)) known to activate capacitative Ca(2+) entry. Unlike carbachol and Tg, EGF (5 nm) elicited a transient [Ca(2+)](i) signal without a plateau phase in the presence of extracellular Ca(2+) and also failed to accelerate Mn(2+) entry. Repletion of extracellular Ca(2+) to cells stimulated with EGF in the absence of Ca(2+) elicited an increase in [Ca(2+)](i), indicating that EGF indeed stimulates Ca(2+) influx. However, the influx was activated at lower EGF concentrations than those required to stimulate Ca(2+) release. Interestingly, the phospholipase C inhibitor completely inhibited Ca(2+) release induced by both EGF and carbachol and also reduced Ca(2+) influx responsive to carbachol but had no effect on Ca(2+) influx induced by EGF. EGF-induced Ca(2+) influx was potentiated by low concentrations (5 ng/ml) of oligomycin, a mitochondrial inhibitor that blocks capacitative Ca(2+) influx in other systems. Transient expression of the hTRPC3 protein enhanced Ca(2+) influx responsive to carbachol but did not increase EGF-activated Ca(2+) influx. Both EGF and carbachol depleted internal Ca(2+) stores. Our results demonstrate that EGF-induced Ca(2+) release from internal stores does not activate capacitative Ca(2+) influx. Rather, EGF stimulates Ca(2+) influx via a mechanism distinct from capacitative Ca(2+) influx induced by carbachol and Tg.

Introduction: MicroRNAs (miRNAs) play important roles in nearly all cellular physiological and pathological pathways. Dysregulated miRNAs have been shown to contribute to the tumorigenesis of most cancers, including lung cancer. We aim to modulate miRNA activity as a potential therapeutic avenue either alone or in combination with other drugs. Methods: A high throughput screen with a library of 1,239 miRNA mimics was performed in NSCLC cells to identify miRNAs that both inhibit cancer cell growth and sensitize cells to microtubule-targeting agents (MTAs). The functions of candidate miRNA(s) were validated both in vitro and in vivo. Results: miR-195 causes G1 phase arrest by targeting CCND3. miR-195 induces apoptosis and senescence and represses migration and invasion of NSCLC cells by targeting BIRC5. Conversely, over-expression of CCND3 compromises the inhibition of cell growth by miR-195; over-expression of BIRC5 abrogates the inhibition of cell growth, migration and invasion by miR-195. miR-195 also synergizes with MTAs paclitaxel and eribulin to inhibit the growth of NSCLC cells by regulating CHEK1. Lung tumors with miR-195 over-expression grow significantly slower and respond better to eribulin treatment than tumors in control animals. Induction of miR-195 expression after tumor initiation slows tumor growth and that combination of doxycycline with eribulin shows stronger inhibition on tumor growth than eribulin. In addition, miR-195 inhibits lung tumor metastasis. Analysis of miRNA and gene expression profiles from TCGA shows that miR-195 expression is negatively correlated with that of BIRC5 and CHEK1. Clinical data from TCGA show that miR-195 is significantly down-regulated in lung tumors compared to adjacent normal tissues and that its down-regulation in lung cancer patients is associated with worse survival. TCGA data also show that BIRC5 and CHEK1 are significantly up-regulated in lung tumors compared to adjacent normal tissues and that their up-regulation is associated with worse survival. Conclusions: We report the identification of miR-195 as a tumor suppressor and chemotherapy sensitizer in NSCLC mediated by its repression of CCND3, BIRC5 and CHEK1. Mouse xenografts with miR-195 over-expression or induced miR-195 expression indicate that tumors with high miR-195 expression are more sensitive to drug treatment and that induction of miR-195 in tumors represses tumor growth and sensitizes tumors to eribulin treatment. These results highlight the potential use of miR-195 expression level as a biomarker to predict patient response to MTAs and of miR-195 mimics as a therapeutic agent or adjuvant to chemotherapy. This project was supported by NIH R01 CA129632 and CPRIT Training Grant RP140105. Citation Format: Xiaojie Yu, Xiuye Ma, Yiqiang Zhang, Zhenze Zhao, Liqin Du, Alexander Pertsemlidis. Therapeutic potential of miR-195 in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5444. doi:10.1158/1538-7445.AM2017-5444

Introduction: Neuroblastoma (NB) is the most common cancer in infants and the most common extracranial solid tumor in children, accounting for 15% of all childhood cancer deaths. The overall prognosis for those with high-risk or relapsed disease remains poor despite the standard therapies of surgery, radiation, and high dose chemotherapy. We have previously shown that altering levels of specific microRNAs (miRNAs) can improve cellular response to microtubule-targeting agents (MTAs) and that exposure to MTAs results in dramatic changes in microRNA expression. Methods: Libraries of chemically synthesized ncRNA mimics and inhibitors, including both miRNAs and lncRNAs, are screened to identify ncRNAs that regulate neuroblastoma cell viability or sensitize neuroblastoma cells to specific microtubule-targeting agents. Candidate targets are validated using qRT-PCR, protein quantification, and luciferase reporter assays. The response of neuroblastoma cells to perturbations in candidate ncRNA levels is assessed through flow cytometric analysis of cell cycle phase distribution, density and anisotropy of microtubule bundles, and through colony formation and caspase activation assays, and validated in mouse xenograft models. Networks of miRNAs, mRNAs and lncRNAs are derived from combining complementary cellular response with potential regulatory interactions. Results: We previously reported the identification of miRNAs and lncRNAs that significantly decrease or increase neuroblastoma cell viability. Here, we extend those observations to drug response. We first demonstrate that the microtubule-targeting agent eribulin has both short-term and long-term effects, as reflected by a significant alteration in microtubule structure in response to high doses for short times and arrest at the G(2)/M phase of the cell cycle in response to low doses for long times. We next show that altering intracellular levels of ncRNAs that sensitize cells to eribulin both alters microtubule density and anisotropy and recapitulates the effect of eribulin treatment. Finally, we show that complementarity of effect between miRNAs and lncRNAs is accompanied by potential regulatory interaction. Conclusions: Taken together, our results suggest that the response of neuroblastoma cells to eribulin is mediated by a regulatory network that includes different coding and non-coding RNA species. While these RNAs may have intrinsic value as biomarkers or therapeutic agents, either individually or in combination, the vulnerabilities that they uncover may be exploited with pathway-specific perturbations. This project was supported by NIH R01 CA129632 and CPRIT Training Grant RP140105. Note: This abstract was not presented at the meeting. Citation Format: Yiqiang Zhang, Xiuye Ma, Xiaojie Yu, Harsh Patolia, Zhenze Zhao, Liqin Du, Alexander Pertsemlidis. ncRNA regulation of eribulin response in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3506. doi:10.1158/1538-7445.AM2017-3506

We previously reported that cAMP inhibits autocrine IGF-I gene expression in rat C6 glioma cells. In this study we examined the influence of cAMP on IGF-binding protein gene expression in C6 cells. cAMP potently inhibited IGF-binding protein-3 mRNA and, to a lesser extent, IGF-binding protein-4 mRNA and transiently stimulated IGF-binding protein-5 mRNA. The changes in secreted IGF-binding proteins whose molecular weights were consistent with IGF-binding protein-3 and -5 correlated with those of mRNA levels. cAMP decreased the IGF-binding protein-3 mRNA half-life, but did not alter IGF-binding protein-4 and -5 mRNA half-lives. An IGF-binding protein-5 promoter/luciferase fusion construct containing 888 bp of 5'-flanking sequence and the first 114 bp of exon 1 sequence was stimulated by cAMP after 24 h by approximately 2-fold in transient transfection assays. 5'- or 3'-deletion to -33 or +10 (the transcription start site was designated as +1), respectively, did not alter the increase caused by cAMP. Site-directed mutagenesis of the region from -14 to -5 led to a loss of the ability of the IGF-binding protein-5 promoter to respond to cAMP. H89, a cell-permeable protein kinase A inhibitor, did not alter the regulation of IGF-binding protein mRNAs in response to cAMP.

The disabled homolog 2 (DAB2) gene was recently identified as a tumor suppressor gene with its expression downregulated in multiple cancer types. The role of DAB2 in lung tumorigenesis, however, is not fully characterized, and the mechanisms of DAB2 dysregulation in lung cancer are not defined. Here we show that low DAB2 levels in lung tumor specimens are significantly correlated with poor patient survival, and that DAB2 overexpression significantly inhibits cell growth in cultured lung cancer cells, indicating its potent tumor suppressor function. We next identify that microRNA miR-93 functions as a potent repressor of DAB2 expression by directly targeting the 3'UTR of the DAB2 mRNA. Using in vitro and in vivo approaches, we demonstrate that miR-93 overexpression has an important role in promoting lung cancer cell growth, and that its oncogenic function is primarily mediated by downregulating DAB2 expression. Our clinical investigations further indicate that high tumor levels of miR-93 are correlated with poor survival of lung cancer patients. The correlations of both low DAB2 and high miR-93 expression levels with poor patient survival strongly support the critical role of the miR-93/DAB2 pathway in determining lung cancer progression.

MicroRNAs (miRNAs) play important roles in nearly all cellular physiological and pathological pathways. Dysregulated miRNAs have been shown to contribute to tumorigenesis of many cancers, including lung cancer. The let-7 miRNA family, among the first miRNAs discovered, suppresses the growth of non-small cell lung cancer (NSCLC). Other miRNAs have been shown to regulate the response of cancer cells to anticancer agents, such as the microtubule-targeting agent (MTA) paclitaxel. These findings highlight the potential application of miRNAs as the next generation of therapeutic agents, either alone or in combination with chemotherapy. A high throughput screen was performed in NSCLC cells. miRNAs that both inhibited NSCLC cell growth and sensitized NSCLC cells to paclitaxel were validated by short- and long-term assays for cell viability. The targets of miRNAs were identified by expression profiling following miRNA transfection, validated by luciferase reporter assay, and confirmed by functional assays. We show that miR-195 causes G1 phase arrest of NSCLC cells by targeting CCND3, and leads to apoptosis and reduction of cell migration and invasion by targeting BIRC5. Conversely, the inhibition of cell growth by miR-195 is compromised by overexpressing CCND3 or BIRC5; the inhibition of cell migration and invasion by miR-195 is abrogated by overexpressing BIRC5. We also show that miR-195 synergizes with MTAs paclitaxel and eribulin to inhibit the growth of NSCLC cells by regulating CHEK1. Analysis of miRNA and gene expression profiles from The Cancer Genome Atlas (TCGA) shows that miR-195 expression is negatively correlated with BIRC5 and CHEK1 but not CCND3. Clinical data from TCGA show that miR-195 is significantly down-regulated in lung tumors compared to adjacent normal tissues and that its down-regulation in lung cancer patients is associated with worse survival. The TCGA data also show that BIRC5 and CHEK1 are significantly up-regulated in lung tumors and that their up-regulation is associated with worse survival. CCND3, however, is slightly downregulated in lung tumors and its expression is not correlated with patient survival. Intriguingly, we also show that miR-195 up-regulates the oncogene MYC, inhibition of which sensitizes NSCLC cells to miR-195. We show that Myc, reported to transcriptionally repress let-7c, also represses miR-195, and further identify a feedback loop between let-7c and miR-195 in which each inhibits the other. We identify miR-195 as a tumor suppressor and chemotherapy sensitizer in NSCLC mediated by its repression of CCND3, BIRC5 and CHEK1. We also uncover a feedback network between miR-195, Myc and let-7c. The ability of miR-195 and Myc to regulate each other indicates a mechanism by which NSCLC cells can become more sensitive to miR-195. The mutual inhibition between miR-195 and let-7c suggests an interesting regulatory loop between cancer-related miRNAs that deserves further study. Citation Format: Xiaojie Yu, Zhenze Zhao, Xiuye Ma, Liqin Du, Alexander Pertsemlidis. miR-195 represses the tumorigenesis of non-small cell lung cancer and synergizes with microtubule targeting agents. [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 1063.

We previously showed that exogenous insulin-like growth factor-I (IGF-I) and bone morphogenetic protein-7 (BMP-7) synergistically stimulated osteoblast differentiation in fetal rat calvaria (FRC) cells. We have now shown that BMP-7 alone and the BMP-7 and IGF-I combination synergistically stimulated protein kinase D (PKD) phosphorylation at Ser744/748 and Ser916. Transfection of FRC cells with a constitutively active PKD stimulated marker expression, while transfection with a catalytically inactive PKD did not. Moreover, G#x00F6;6976, which inhibits protein kinase C (PKC)#x03B1; and#x03B2;1, blocked PKD phosphorylation and the synergistic action of the BMP-7 and IGF-I combination on osteoblast differentiation, whereas G#x00F6;6983, which inhibits PKC#x03B1;,#x03B2;,#x03B3;,#x03B4;, and#x03B6;, did not. Our results suggest that the FRC cell differentiation induced by BMP-7 and the BMP-7 and IGF-I combination requires stimulation of PKD activity. Our results are consistent with a novel mechanism in which combined BMP-7 and IGF-I signaling activates upstream novel PKC(s), which then phosphorylates and activates PKD, leading to enhanced osteoblast differentiation.

It has been shown that store-operated Ca2+influx (SOC) plays critical roles in the activation of endothelial nitric oxide (NO) synthase (eNOS) and generation of NO in endothelial cells. Recent studies indicate stromal interaction molecule 1 (STIM1) is the molecule responsible for SOC activation following Ca2+depletion in the ER. Retinoic acids (RA) have beneficial effects in the treatment of renal diseases. The mechanism of the RA action is still largely unknown. In the current study, we used primary cultured rat mesangial cells to examine the effect of RA on SOC and STIM1. In these cells, BK caused concentration-dependent [Ca2+]imobilization. Treatment of the cells with RA, while it had no effect on the initial peak, reduced the plateau phase of BK-mediated [Ca2+]iresponse, indicating the inhibition of SOC by RA. The level of STIM1 protein but not mRNA in RA-treated cells was significantly reduced. RA treatment did not affect TGF-β-mediated gradual Ca2+influx which occurred by superoxide anion-mediated mechanism, indicating RA treatment specifically inhibited SOC in mesangial cells. RT-PCR and Western blot analysis demonstrated that eNOS was expressed in rat mesangial cells grown in media containing 11 and 30 but not 5.5 mM glucose. Downregulation of STIM1 protein and BK-induced SOC by RA treatment or STIM1 dsRNA were associated with abolished NO production. The 26S proteasome inhibitor lactacystin blocked the RA-mediated downregulation of BK-induced SOC, suggesting that ubiquitin-proteasome pathway may be involved in RA-mediated STIM1 protein downregulation in rat mesangial cells. Our data suggest that glucose-induced eNOS expression and NO production in mesangial cells may contribute to hyperfiltration in diabetes and RA may exert beneficial effects by downregulation of STIM1 and SOC in mesangial cells.

In a variety of disorders, overaccumulation of lipid in nonadipose tissues, including the heart, skeletal muscle, kidney, and liver, is associated with deterioration of normal organ function, and is accompanied by excessive plasma and cellular levels of free fatty acids (FA). Increased concentrations of FA may lead to defects in mitochondrial function found in diverse diseases. One of the most important regulators of mitochondrial function is mitochondrial Ca2+([Ca2+]m), which fluctuates in coordination with intracellular Ca2+([Ca2+]i). Polyunsaturated FA (PUFA) have been shown to cause [Ca2+]imobilization albeit by unknown mechanisms. We have found that PUFA but not monounsaturated or saturated FA cause [Ca2+]imobilization in NT2 human teratocarcinoma cells. Unlike the [Ca2+]iresponse to the muscarinic G protein-coupled receptor agonist carbachol, PUFA-mediated [Ca2+]imobilization in NT2 cells is independent of phospholipase C and inositol-1,4,5-trisphospate (IP3) receptor activation, as well as IP3-sensitive internal Ca2+stores. Furthermore, PUFA-mediated [Ca2+]imobilization is inhibited by the mitochondria uncoupler carboxyl cyanide m-chlorophenylhydrozone. Direct measurements of [Ca2+]mwith X-rhod-1 and45Ca2+indicate that PUFA induce Ca2+efflux from mitochondria. Further studies show that ruthenium red, an inhibitor of the mitochondrial Ca2+uniporter, blocks PUFA-induced Ca2+efflux from mitochondria, whereas inhibitors of the mitochondrial permeability transition pore cyclosporin A and bongkrekic acid have no effect. Thus PUFA-gated Ca2+release from mitochondria, possibly via the Ca2+uniporter, appears to be the underlying mechanism for PUFA-induced [Ca2+]imobilization in NT2 cells.

C3H/HeJ (C3H) mice exhibit 30-40% higher serum IGF-I than do C57BL/6J (B6) mice, in association with increased bone mineral density and strength. These differences are inherited and thus provide a model for determining molecular mechanisms for genetic variation of serum IGF-I and downstream actions. We now report that increased serum IGF-I in C3H mice is associated with increased transcription from the minor exon 2 promoter in liver from female and male mice. The increase in hepatic IGF-I gene expression caused by increased abundance of IGF-I mRNA transcribed from the exon 2 promoter can quantitatively account for the increased serum IGF-I in C3H mice. Also, levels of both Ea and Eb IGF-I mRNAs are increased in livers of male C3H mice. Fasting lowered serum IGF-I and liver IGF-I mRNA levels in female mice of both strains. However, serum IGF-I and liver IGF-I mRNA levels remained higher in fasted C3H mice compared with fasted B6 mice. Levels of IGF-I transcripts initiated from exon 2 are also significantly increased in skeletal muscle, fat, ovaries, and kidneys of C3H mice. IGF binding protein (IGFBP)-5 mRNA levels are significantly higher in muscle and fat of C3H mice than in B6 mice. Levels of exon 1-containing transcripts are increased in whole femurs of male and female C3H mice. We conclude that increased transcription of the IGF-I gene occurs in a promoter- and tissue-specific manner in C3H mice. The increased IGF binding protein-5 mRNA levels in fat and muscle suggest that IGF-I signaling is increased in these tissues in C3H mice.

We previously reported that reduction of autocrine IGF-I by polyinosinic-polycytidylic acid [poly(IC)] was permissive for the poly(IC)-mediated decrease in C6 rat glioma cell number. We now report that poly(IC) caused a block in G(1) to S transition in confluent C6 cultures, whereas in subconfluent cultures, poly(IC) decreased the percentage of cells in the G(2)/M phase. Addition of IGF-I to poly(IC)-treated cells decreased the percentage of cells in G(0)/G(1) phase and increased the percentage of cells in G(2)/M phase in confluent and subconfluent C6 cultures, indicating the reversal of cell cycle blocks. Inhibition of protein kinase R (PKR) activation partially prevented the poly(IC)-mediated cytostasis of C6 cells. Poly(IC) induced interferon-alpha in C6 cells. Both IGF-I and a blocking antibody against type I interferon (IFN) prevented the increase in PKR levels and the decrease in cell proliferation caused by poly(IC). We conclude that poly(IC) induces IFN, which mediates the cytostatic effect of poly(IC) on C6 cells at least in part through PKR. IGF-I prevents IFN from inducing PKR, thus explaining the ability of IGF-I to reverse the cell cycle blocks and the decreased C6 proliferation caused by poly(IC).

Introduction. microRNAs (miRNAs) are a family of small non-coding RNAs that post-transcriptionally regulate gene expression mainly by decreasing mRNA levels of target genes. Recently, numerous studies have shown the important roles of miRNAs in regulating various cellular physiological and pathological pathways including cell differentiation, proliferation and even tumorigenesis. A large number of miRNAs are indicated to be involved in multiple pathways that are critical for lung cancer, which is the leading cause of adult cancer deaths. Intriguingly, some miRNAs sensitize lung cancer to microtubule-targeting agents, such as paclitaxel. All of these highlight the potential application of miRNA-targeted oligos as next-generation therapeutic agents. Aims and Methods. We aim to identify novel functional miRNAs in lung cancer. Using a high-throughput screening platform containing a library of chemically synthesized miRNAs, we systematically identify miRNAs that reduce lung cancer cell survival or sensitize cancer cells to paclitaxel. The top candidate miRNAs are validated and their mechanisms are further investigated. Results and Conclusions. We show that miR-195 significantly inhibits the viability of lung cancer cells in vitro, and that transfection of miR-195 into cancer cells results in G1 phase arrest and decreased cell invasion. Intriguingly, miR-195 does not show strong inhibitory effects on viability of immortalized human bronchial epithelial cells (HBECs). Analysis of miRNA expression data from The Cancer Genome Atlas (http://cancergenome.nih.gov) indicates lower expression of miR-195 in lung cancer tumors compared to normal tissues. Merging expression with clinical annotation shows that higher expression of miR-195 in lung cancer patients is associated with longer survival. These results indicate that miR-195 is a tumor suppressor miRNA and that miR-195 may be an important factor in determining lung cancer progression and prognosis. This observation is consistent with previous studies that have shown a tumor suppressor function of miR-195 in breast cancer, bladder cancer and hepatocellular carcinoma. Overall, the inhibitory effects on lung cancer cell growth and the correlation between expression and survival indicate the involvement of miR-195 in critical pathways for lung cancer. We have identified additional miRNAs that sensitize lung cancer cells to paclitaxel, and which define a potential regulatory pathway modulating paclitaxel sensitivity. Future studies are needed to identify the targets of these miRNAs, elucidate their mechanisms of action, and clarify how they are regulated in lung cancer cells. Citation Format: Xiaojie Yu, Zhenze Zhao, Xiuye Ma, Liqin Du, Alexander Pertsemlidis. A high-throughput screen identifies microRNAs regulating lung cancer cell survival and response to paclitaxel. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3545. doi:10.1158/1538-7445.AM2014-3545

Introduction. The aim of the study is to characterize the role of LMO1 in tumorigenesis of neuroendocrine lung cancer. LMO1 is a transcription factor that belongs to the LMO protein family containing the cysteine-rich LIM domains. LMO1 has been demonstrated to function as an important oncogene in the development of T-lineage leukemia and lymphoma. A recent study in neuroblastoma suggests that aberrant overexpression of LMO1 may play a critical role in the development of neuroendocrine cancers. The role of LMO1 in neuroendocrine lung cancer, however, was not investigated. Experimental Approaches. By analyzing a large panel of cell lines including lung cancer cell lines and normal lung epithelial cells, we compared the relative expression of LMO1 in different subtypes of lung cancers, and examined the correlation of LMO1 expression levels with neuroendocrine differentiation markers. Using in vitro approaches, we investigated the function of LMO1 in regulating lung cancer cell growth. To investigate the clinical significance of LMO1 dysregulation in lung cancer patients, we analyzed the correlation of LMO1 tumor levels with lung cancer patient survival. Results. We show that LMO1 is significantly overexpressed in small cell lung cancer (SCLC), an aggressive and major subtype of neuroendocrine lung cancers, relative to non-small cell lung cancer (NSCLC) and normal lung cells. In addition, in NSCLC cells, LMO1 mRNA levels are significantly correlated with expression of neuroendocrine differentiation markers. Functional in vitro investigations indicate that LMO1 overexpression significantly promotes growth of cultured lung cancer cells, suggesting its oncogenic function in lung cancer. More strikingly, our investigations of two independent lung cancer patient populations indicate that high tumor LMO1 mRNA level is an independent predictor of poor patient survival. Conclusions. Altogether, our findings strongly suggest that LMO1 is a neuroendocrine-specific oncogene in lung cancer that plays an important role in determining the cancer aggressiveness. Further studies are certainly warranted to define the mechanisms underlying the oncogenic function of LMO1 in neuroendocrine lung cancer and to further evaluate the clinical significance of LMO1 as a diagnostic and prognostic marker for neuroendocrine lung cancers in prospective studies. Citation Format: Zhenze Zhao, Xiuye Ma, Xiaojie Yu, Tzu-Hung Hsiao, Yidong Chen, Milind Suraokar, Ignacio Wistuba, John D. Minna, Alexander Pertsemlidis, Liqin Du. LMO1 is a novel oncogene in neuroendocrine lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 466. doi:10.1158/1538-7445.AM2014-466

Introduction. The Disabled homolog 2 (DAB2) gene is located on human chromosome 5p13 and expresses a mitogen-responsive phosphoprotein. Previous studies have indicated that DAB2 is a tumor suppressor gene and is down-regulated in multiple cancer types, suggesting that repressed DAB2 expression plays an important role in oncogenesis. Loss of Dab2 protein expression has also been observed in lung cancer. The molecular and clinical consequences of loss of Dab2 expression in lung cancers, however, have not been fully evaluated, and the mechanisms of its dysregulation in cancers need to be defined. Aims and Methods. We aim to determine the role of DAB2 in lung tumorigenesis, and the role of microRNAs (miRNAs) in regulating Dab2 expression in lung cancer cells. Using in silico and in vitro approaches, we identified DAB2 as a direct target of miR-93. Using in vitro approaches, we investigated the function of Dab2 and miR-93 in regulating lung cancer cell growth. To investigate the clinical significance of DAB2 and miR-93 dysregulation in lung cancer patients, we examined the correlation of miR-93 and DAB2 tumor levels with lung cancer patient survival, and investigated the correlation between miR-93 and DAB2 expression in lung tumor specimens. Results and Conclusions. We show that Dab2 overexpression significantly arrests cell growth in lung cancer cells in vitro, and that low DAB2 mRNA levels in human lung tumor specimens are significantly correlated with poor patient survival. These results indicate that Dab2 is a potent tumor suppressor in lung cancer and that DAB2 levels may be an important factor in determining lung cancer progression and prognosis. We show that miR-93 directly targets the 3’UTR of DAB2 mRNA, and that miR-93 levels are negatively correlated with DAB2 mRNA levels in lung cancer specimens. We also show that high miR-93 levels are correlated with poor patient survival. Together these results support the clinical importance of the miR-93/Dab2 regulatory pathway in lung cancer. Overall, our study identifies a novel oncogenic pathway in lung cancer mediated by over-expression of miR-93 and subsequent down-regulation of its target DAB2. The significant correlations of both DAB2 and miR-93 expression with patient survival highlight the critical role of this pathway in lung tumorigenesis. Future studies are needed to investigate whether therapeutic tools targeting this pathway could be developed for lung cancer treatment, and whether this pathway provides a valuable prognostic biomarker for lung cancer patient survival. Citation Format: Liqin Du, Zhenze Zhao, Emily Young, Xiuye Ma, Ignacio Wistuba, Alexander Pertsemlidis. miR-93-directed down-regulation of Dab2 defines a novel oncogenic pathway 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 3096. doi:10.1158/1538-7445.AM2013-3096

Extracellular ATP is an important neurotransmitter that modulates cardiac function by activation of purinergic receptors. In this study, the effect of P 2 purinergic receptor activation on contractions and on [Ca 2+ ] i was investigated in adult rat ventricular myocytes. Fura 2 was used to measure [Ca 2+ ] i , and video edge detection was used to measure contraction. Superfusion of 2-methylthio-adenosine-5′-triphosphate (2-M-S-ATP) over quiescent myocytes induced oscillations in contraction and in [Ca 2+ ] i . The frequency of the oscillatory contractions increased with increasing concentrations of 2-M-S-ATP, but the amplitude of contractions varied from cell to cell and was independent of the concentration of 2-M-S-ATP. During electrical stimulation, activation of purinergic receptors in myocytes potentiated the amplitude of contraction and induced arrhythmias. In populations of quiescent myocytes, the plateau phase of the [Ca 2+ ] i signal evoked by 2-M-S-ATP could be shown to represent summed oscillations in [Ca 2+ ] i in individual cells. Pretreatment of quiescent myocytes with thapsigargin or caffeine reduced or abolished the oscillations in contractions and in [Ca 2+ ] i triggered by 2-M-S-ATP, indicating a dependence of the oscillations on uptake and release of Ca 2+ by the sarcoplasmic reticulum. These data demonstrate the novel phenomenon that activation of purinergic receptors in quiescent myocytes stimulates oscillations in [Ca 2+ ] i and contraction. In electrically stimulated myocytes, activation of purinergic receptors triggers oscillatory contractions and potentiates the amplitude of electrically triggered contractions.

Neuroblastoma, the most common extracranial solid tumor of childhood, arises from neural crest cell precursors that fail to differentiate. Inducing cell differentiation is an important therapeutic strategy for neuroblastoma. We developed a direct functional high-content screen to identify differentiation-inducing microRNAs, in order to develop microRNA-based differentiation therapy for neuroblastoma. We discovered novel microRNAs, and more strikingly, three microRNA seed families that induce neuroblastoma cell differentiation. In addition, we showed that microRNA seed families were overrepresented in the identified group of fourteen differentiation-inducing microRNAs, suggesting that microRNA seed families are functionally more important in neuroblastoma differentiation than microRNAs with unique sequences. We further investigated the differentiation-inducing function of the microRNA-506-3p/microRNA-124-3p seed family, which was the most potent inducer of differentiation. We showed that the differentiation-inducing function of microRNA-506-3p/microRNA-124-3p is mediated, at least partially, by down-regulating expression of their targets CDK4 and STAT3. We further showed that expression of miR-506-3p, but not miR-124-3p, is dramatically upregulated in differentiated neuroblastoma cells, suggesting the important role of endogenous miR-506-3p in differentiation and tumorigenesis. Overall, our functional screen on microRNAs provided the first comprehensive analysis on the involvements of microRNA species in neuroblastoma cell differentiation and identified novel differentiation-inducing microRNAs. Further investigations are certainly warranted to fully characterize the function of the identified microRNAs in order to eventually benefit neuroblastoma therapy.