Knockdown Clones Research Articles

Abstract P6-01-19: Decoding the genetic basis of mammary mineralization and their putative role in promotion of distant metastases

Abstract Background: Breast microcalcifications are the sole early stage diagnostic markers of breast cancer. The association of mineralization (especially type II microcalcifications) with both benign and malignant lesions often leads to unnecessary biopsies. The processes by which these ectopic microcalcifications form are unknown. In the current work, we attempted to explore the possibility of obtaining genes responsible for the formation of microcalcifications in breast cancer cell lines at cellular level and understand their potential involvement in disease progression and distant metastases. Methods:The GEO dataset GSE16795 used in this study contains gene expression profiles of 28 human breast cancer cell lines that were divided into two groups - metastatic and non-metastatic. Gene expression levels of OPN were found to be significantly (p=0.0002) elevated for the metastatic group compared to the non-metastatic group. Hence, the human breast cancer cell lines - metastatic (MDA-MB-231 and SUM 149) and non-metastatic (BT-474 and T47D) from the American Type Culture Collection were cultured and their OPN expression at mRNA and protein levels determined by qRT-PCR and Western blotting were compared. Additionally the same cell lines were cultured in media enriched with an osteogenic cocktail containing 10mM β-glycerophosphate (Sigma, USA) and 50 mg/ml-1 ascorbic acid (Sigma, USA) for induction of microcalcifications.Next, several clones were generated using shRNA knockdown of OPN gene in MDA-MB-231 cells for further study. In vitro studies were conducted to assess the effects of OPN knockdown on the migration and invasion potential of MDA-MB-231 cells using transwell migration assays. Results: The expression of OPN at both mRNA and protein levels are significantly higher for the metastatic cell lines when compared to non-metastatic cell lines. It can also be observed that OPN expression in the cells increases substantially with addition of exogenous phosphates in the form of osteogenic cocktail and thereby indicating that OPN possibly plays a crucial role in mediating formation of microcalcifications in these cells(P<0.001). Metastatic cell line MDA-MB-231 was employed as a model system for further investigation in this study. There is a consistent inhibition of formation of cellular microcalcifications due to the knockdown of gene responsible for OPN, suggesting that OPN gene is directly associated with the formation and regulation of hydroxyapatite formation in MDA-MB-231 breast cancer. Further, the above observation is strengthened by the similarity of the trend of variation in level of mRNA expression of OPN and cellular calcification content across the knockdown clones. OPN knockdown cell lines show reduced cell migration and invasion in the assays compared to the control MDA-MB-231 cells. Conclusion: The knockdown of OPN gene not only reduced the formation of microcalcifications in the cells in response to osteogenic cocktail but also affected their migration and invasion characteristics. The observed dual roles of the OPN gene encourage us to probe further into the possible existence of a direct relationship between microcalcifications and ability to metastasize to distant organs mediated by common genetic factors in the future. Citation Format: Zheng C, Rizwan A, Paidi SK, Yu Z, Barman I, Glunde K. Decoding the genetic basis of mammary mineralization and their putative role in promotion of distant metastases [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-01-19.

TET1 Depletion Induces Aberrant CpG Methylation in Colorectal Cancer Cells.

Aberrant DNA methylation is commonly observed in colorectal cancer (CRC), but the underlying mechanism is not fully understood. 5-hydroxymethylcytosine levels and TET1 expression are both reduced in CRC, while epigenetic silencing of TET1 is reportedly associated with the CpG island methylator phenotype. In the present study, we aimed to clarify the relationship between loss of TET1 and aberrant DNA methylation in CRC. Stable TET1 knockdown clones were established using Colo320DM cells, which express high levels of TET1, and HCT116 cells, which express TET1 at a level similar to that in normal colonic tissue. Infinium HumanMethylation450 BeadChip assays revealed increased levels of 5-methylcytosine at more than 10,000 CpG sites in TET1-depleted Colo320DM cells. Changes in DNA methylation were observed at various positions within the genome, including promoters, gene bodies and intergenic regions, and the altered methylation affected expression of a subset of genes. By contrast, TET1 knockdown did not significantly affect DNA methylation in HCT116 cells. However, TET1 depletion was associated with attenuated effects of 5-aza-2’-deoxycytidine on gene expression profiles in both cell lines. These results suggest that loss of TET1 may induce aberrant DNA methylation and may attenuate the effect of 5-aza-2’-deoxycytidine in CRC cells.

Cannabinoid receptor interacting protein suppresses agonist-driven CB1 receptor internalization and regulates receptor replenishment in an agonist-biased manner.

Cannabinoid receptor interacting protein 1a (CRIP1a) is a CB1 receptor (CB1 R) distal C-terminus-associated protein that modulates CB1 R signaling via G proteins, and CB1 R down-regulation but not desensitization (Blume etal. [2015] Cell Signal., 27, 716-726; Smith etal. [2015] Mol. Pharmacol., 87, 747-765). In this study, we determined the involvement of CRIP1a in CB1 R plasma membrane trafficking. To follow the effects of agonists and antagonists on cell surface CB1 Rs, we utilized the genetically homogeneous cloned neuronal cell line N18TG2, which endogenously expresses both CB1 R and CRIP1a, and exhibits a well-characterized endocannabinoid signaling system. We developed stable CRIP1a-over-expressing and CRIP1a-siRNA-silenced knockdown clones to investigate gene dose effects of CRIP1a on CB1 R plasma membrane expression. Results indicate that CP55940 or WIN55212-2 (10nM, 5 min) reduced cell surface CB1 R by a dynamin- and clathrin-dependent process, and this was attenuated by CRIP1a over-expression. CP55940-mediated cell surface CB1 R loss was followed by a cycloheximide-sensitive recovery of surface receptors (30-120min), suggesting the requirement for new protein synthesis. In contrast, WIN55212-2-mediated cell surface CB1 Rs recovered only in CRIP1a knockdown cells. Changes in CRIP1a expression levels did not affect a transient rimonabant (10nM)-mediated increase in cell surface CB1 Rs, which is postulated to be as a result of rimonabant effects on 'non-agonist-driven' internalization. These studies demonstrate a novel role for CRIP1a in agonist-driven CB1 R cell surface regulation postulated to occur by two mechanisms: 1) attenuating internalization that is agonist-mediated, but not that in the absence of exogenous agonists, and 2) biased agonist-dependent trafficking of de novo synthesized receptor to the cell surface.

Abstract 3368: TIMP-1 and angiogenesis: A role for CDH5 and eNOS

Abstract Tissue inhibitors of metalloproteinases (TIMP) have emerged as diverse molecules with novel roles in apoptosis, angiogenesis and metastasis. TIMP-1, a well-documented inhibitor of apoptosis, has been shown to inhibit or promote angiogenesis. In recent years, a strong association has also been demonstrated between high levels of TIMP-1 and poor prognosis in many cancers. It has been recognized as a multifunctional protein affecting cell growth and angiogenesis. Conflicting studies have shown it to function either as a negative or a positive regulator of angiogenesis. In earlier studies, we have documented that lung adenocarcinoma cell line H2009, when transfected to overexpress TIMP-1 and injected into nude mice resulted in larger, more aggressive tumors with increased microvessel density, which was supported by in vitro angiogenesis assays whereby enhanced capillary network formation was seen. We have also shown that TIMP-1 expression levels can be correlated with KRAS independency in non-small-cell lung carcinoma (NSCLC) cell lines harboring KRAS mutations. The present study was undertaken to address the role of TIMP-1 in angiogenesis in the context of KRAS in NSCLC cell lines. KRAS dependent cell line H2009 and its TIMP-1 overexpressing clone HB1 and a KRAS independent cell line A549 and its TIMP-1 knockdown (KD) clone SH3 were examined by angiogenesis specific PCR array. Comparison of the angiogenesis associated profiles of these cell lines identified a marked increase in vascular endothelial cadherin CDH5 in TIMP-1 over-expressing clone HB1. KRAS independent A549 cells expressed high level of CDH5, however its TIMP-1 KD clone exhibited a marked decrease in CDH5 level. A similar profile was identified for endothelial nitric oxide synthase (eNOS/NOS3). CDH5 is an indispensable element of normal vascular development and maintenance, and any perturbations of normal levels will impact vessel sprouting and growth. eNOS is primarily active through production of nitric oxide, maintaining vascular tone as well as expressing anti-proliferative and antithrombotic properties. We confirmed our earlier observation that serum free conditioned media (SFCM) from TIMP-1 overexpressing HBI cells caused increased and more complex network formation. SFCM from A549 TIMP-1 KD SH3 clone showed a marked inhibition of endothelial network formation compared to SFCM from A549 cells. To confirm that TIMP-1 was responsible for the changes observed we purified secretory TIMP-1 by combined fast protein liquid chromatography and gel filtration from another high TIMP-1 producing NSCLC cell line (H460) and also observed more capillary network formation in human umbilical vein endothelial cells. Our studies suggest that TIMP-1 modulates angiogenesis by impacting CDH5 and eNOS positively. These results define an important function of TIMP-1 in angiogenesis and provide additional therapeutic targets for managing NSCLC. Citation Format: Sampa Ghoshal-Gupta, Ian A. Coe, Ammar Kutiyanawalla, Byung R. Lee, Amyn M. Rojiani, Mumtaz V. Rojiani. TIMP-1 and angiogenesis: A role for CDH5 and eNOS. [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 3368.

Dengue Virus Nonstructural Protein 1 Induces Vascular Leakage through Macrophage Migration Inhibitory Factor and Autophagy.

Dengue virus (DENV) is the most common mosquito-borne flavivirus; it can either cause mild dengue fever or the more severe dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). One of the characteristic features of DHF/DSS is vascular leakage; although DENV nonstructural protein 1 (NS1) has been proved to induce vascular leakage after binding to Toll-like receptor 4, the down-stream mechanism has not yet been fully understood. In the sera of DENV-infected patients, the concentrations of DENV NS1 and inflammatory cytokine macrophage migration inhibitory factor (MIF) are positively correlated with disease severity, but whether DENV NS1 induces vascular leakage through MIF secretion remains unknown. We demonstrated that recombinant NS1 induced vascular leakage and MIF secretion both in human endothelial cell line HMEC-1 and in mice. Furthermore, these phenomena were inhibited in the presence of anti-NS1 antibodies both in vitro and in vivo. DENV NS1 also induced LC3-I to LC3-II conversion and p62 degradation in endothelial cell line, which indicated the formation of autophagy. To clarify whether MIF or autophagy mediated DENV NS1-induced vascular leakage, various inhibitors were applied. The results showed that DENV NS1-induced vascular leakage and VE-cadherin disarray were blocked in the presence of MIF inhibitors, anti-MIF-antibodies or autophagy inhibitors. An Atg5 knockdown clone further confirmed that autophagy formation of endothelial cells was required in NS1-induced vascular leakage. Furthermore, DENV NS1-induced LC3 puncta were also decreased in the presence of MIF inhibitors, indicating that MIF mediated DENV NS1-induced autophagy. Taken together, the results suggest a potential mechanism of DENV-induced vascular leakage and provide possible therapeutic targets against DHF/DSS.

Stable Toll-Like Receptor 10 Knockdown in THP-1 Cells Reduces TLR-Ligand-Induced Proinflammatory Cytokine Expression

Toll-like receptor 10 (TLR10) is the only orphan receptor whose natural ligand and function are unknown among the 10 human TLRs. In this study, to test whether TLR10 recognizes some known TLR ligands, we established a stable TLR10 knockdown human monocytic cell line THP-1 using TLR10 short hairpin RNA lentiviral particle and puromycin selection. Among 60 TLR10 knockdown clones that were derived from each single transduced cell, six clones were randomly selected, and then one of those clones, named E7, was chosen for the functional study. E7 exhibited approximately 50% inhibition of TLR10 mRNA and protein expression. Of all the TLRs, only the expression of TLR10 changed significantly in this cell line. Additionally, phorbol 12-myristate 13-acetate-induced macrophage differentiation of TLR10 knockdown cells was not affected in the knockdown cells. When exposed to TLR ligands, such as synthetic diacylated lipoprotein (FSL-1), lipopolysaccharide (LPS), and flagellin, significant induction of proinflammatory cytokine gene expression including Interleukin-8 (IL-8), Interleukin-1 beta (IL-1β), Tumor necrosis factor-alpha (TNF-α) and Chemokine (C–C Motif) Ligand 20 (CCL20) expression, was found in the control THP-1 cells, whereas the TLR10 knockdown cells exhibited a significant reduction in the expression of IL-8, IL-1β, and CCL20. TNF-α was the only cytokine for which the expression did not decrease in the TLR10 knockdown cells from that measured in the control cells. Analysis of putative binding sites for transcription factors using a binding-site-prediction program revealed that the TNF-α promoter does not have putative binding sites for AP-1 or c-Jun, comprising a major transcription factor along with NF-κB for TLR signaling. Our results suggest that TLR10 is involved in the recognition of FSL-1, LPS, and flagellin and TLR-ligand-induced expression of TNF-α does not depend on TLR10.

Phosphorylation-Dependent Regulation of the DNA Damage Response of Adaptor Protein KIBRA in Cancer Cells.

Multifunctional adaptor proteins encompassing various protein-protein interaction domains play a central role in the DNA damage response pathway. In this report, we show that KIBRA is a physiologically interacting reversible substrate of ataxia telangiectasia mutated (ATM) kinase. We identified the site of phosphorylation in KIBRA as threonine 1006, which is embedded within the serine/threonine (S/T) Q consensus motif, by site-directed mutagenesis, and we further confirmed the same with a phospho-(S/T) Q motif-specific antibody. Results from DNA repair functional assays such as the γ-H2AX assay, pulsed-field gel electrophoresis (PFGE), Comet assay, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, and clonogenic cell survival assay using stable overexpression clones of wild-type (wt.) KIBRA and active (T1006E) and inactive (T1006A) KIBRA phosphorylation mutants showed that T1006 phosphorylation on KIBRA is essential for optimal DNA double-strand break repair in cancer cells. Further, results from stable retroviral short hairpin RNA-mediated knockdown (KD) clones of KIBRA and KIBRA knockout (KO) model cells generated by a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system showed that depleting KIBRA levels compromised the DNA repair functions in cancer cells upon inducing DNA damage. All these phenotypic events were reversed upon reconstitution of KIBRA into cells lacking KIBRA knock-in (KI) model cells. All these results point to the fact that phosphorylated KIBRA might be functioning as a scaffolding protein/adaptor protein facilitating the platform for further recruitment of other DNA damage response factors. In summary, these data demonstrate the imperative functional role of KIBRAper se(KIBRA phosphorylation at T1006 site as a molecular switch that regulates the DNA damage response, possibly via the nonhomologous end joining [NHEJ] pathway), suggesting that KIBRA could be a potential therapeutic target for modulating chemoresistance in cancer cells.

Abstract B26: Identification of genes that predict response to paclitaxel in breast cancer using an in vivo genome-wide knockdown screen

Abstract Treatment decisions for breast cancer are based upon stage, tumor grade and hormone receptor status, and can include surgical resection, hormone receptor antagonists, radiation, and chemotherapy (e.g. paclitaxel). Breast cancer treatment success depends upon avoidance of chemotherapy resistance (i.e. achieving complete response) and prevention of both over- and under-treatment. Increased understanding of the genes which cause resistance and sensitivity to currently used drugs would lead to development of more effective therapeutic strategies that are specifically tailored to patient groups based on molecular profiling of their tumors (i.e. personalized medicine). Being able to identify the genes which when expressed in a tumor predict sensitivity or resistance to treatment prior to administration of paclitaxel would improve treatment efficacy and patient survival. We performed an in vivo shRNA genome-wide screen with MDA-MB-231 tumors treated with paclitaxel for the purpose of identifying genes which determine breast cancer response to paclitaxel. Completion of 6 replicates of the in vivo screen identified 26 putative paclitaxel sensitivity genes and 14 putative paclitaxel resistance genes (e.g. BCL6) for breast cancer. Screen-identified putative paclitaxel resistance were verified by individual knockdown clone generation and comparison of their sensitivity to paclitaxel-induced decreased cell proliferation, cell-cycle arrest, and apoptosis to a shRNA scramble control clone. Upon individual knockdown of the putative resistance genes (e.g. BCL6), MDA-MB-231 cells were more sensitive to paclitaxel and demonstrated increased apoptosis and decreased paclitaxel IC50 concentrations. Finally, expression of a preliminary gene signature generated from the screen-identified hits was tested for its ability to predict response to paclitaxel in two archived patient data sets. The preliminary gene signature predicted response to paclitaxel in the datasets with an accuracy ranging from 70 to 100%. Further confirmation experiments of the remaining potential resistance and sensitivity genes will help to generate a more robust genetic profile which can be used to identify candidate breast cancer patients who would most benefit from paclitaxel treatment as opposed to treatment with other drugs. Citation Format: Mohammad Sultan, Thomas Tan Huynh, Margaret Lois Thomas, Krysta Mila Coyle, Carman A. Giacomantonio, Paola Marcato. Identification of genes that predict response to paclitaxel in breast cancer using an in vivo genome-wide knockdown screen. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B26.

Abstract A30: Frizzled-7 (FZD7)-mediated non-canonical Wnt-Planar Cell Polarity (PCP) signalling pathway as a novel molecular driver for the C5/Proliferative/Stem-A molecular subtype of ovarian cancer.

Abstract Ovarian cancer (OC), in particular high grade serous carcinoma (HGSC), has been shown to exhibit diverse molecular heterogeneity based on gene expression profiling by the Australian and the TCGA cohorts. This molecular heterogeneity has been demonstrated to be very robust and reproducible by a large-scale meta-analysis study consisting of 1,538 samples from our group. At least 5 distinct gene-expression based molecular subtypes (GEMS) of EOC have been identified. The C5 subtype from the Tothill dataset corresponds to the Proliferative subtype from the TCGA dataset and the Stem-A subtype from the 1,538 meta-analysis dataset. These GEMS have been correlated with patient survival. The C5/Proliferative/Stem-A GEMS is associated with poorer survival outcomes. From our own study, Stem-A OC cells tend to display preferential chemosensitivity towards microtubule depolymerizing agents affecting the plus-end dynamics of microtubule such as vincristine and vinorelbine. Interestingly, Stem-A as well as non-Stem-A cells are equally sensitive to microtubule stabilizing agents such as paclitaxel. The biology of this C5/Proliferative/Stem-A-subtyped OC is still elusive and requires in-depth study. Here, we demonstrate that a Wnt receptor Frizzled-7 (FZD7) is crucial for the aggressiveness of the C5/Proliferative/Stem-A GEMS. Silencing FZD7 in the Stem-A-subtyped OC induces decrease in cell proliferation and cell cycle arrest. Silencing FZD7 also induced compaction of OC cell colony and strengthening of actin cytoskeleton at cell-cell junctions. Stable FZD7 knockdown clones further demonstrate significant decrease in anoikis resistance, spheroid forming ability, and tumorigenesis in xenografts. We further show that these functional changes acts via the Wnt-planar cell polarity (PCP) pathway downstream of FZD7. A subset of PCP pathway genes is also enriched in Stem-A tumors. Transient silencing of three downstream PCP pathway genes, CELSR3, PRICKLE4, PTK7, gave rise to similar functional phenotypes as silencing FZD7. These FZD7-PCP silenced Stem-A OC cells also demonstrate enhanced sensitivity to the microtubule depolymerizing agent vinorelbine. We conclude that the FZD7-PCP pathway drives the aggressiveness of the C5/Proliferative/Stem-A-subtyped OC and can be potentially utilized as the marker to identify the Stem-A subgroup. Molecularly stratifying OC patients and targeting C5/Proliferative/Stem-A subtype with therapeutics against FZD7-PCP pathway would be a novel strategy for OC. Citation Format: Mohammad Asad, Meng Kang Wong, Tuan Zea Tan, David Virshup, Jean Paul Thiery, Ruby Yun-Ju Huang. Frizzled-7 (FZD7)-mediated non-canonical Wnt-Planar Cell Polarity (PCP) signalling pathway as a novel molecular driver for the C5/Proliferative/Stem-A molecular subtype of ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A30.

Post-transcriptional regulation of Rbm5 expression in undifferentiated H9c2 myoblasts

We previously examined the expression of Rbm5 during myoblast differentiation and found significantly more protein in the early stages of skeletal myoblast differentiation than during the later stages. We decided to determine if this elevated level was necessary for differentiation. Our hypothesis was that if high levels of Rbm5 protein expression were necessary for the initiation of skeletal myoblast differentiation, then inhibition of expression would prevent differentiation. Our long-term objective is to inhibit Rbm5 expression and examine the effect on H9c2 differentiation. Towards this end, stable knockdown clones and transient knockdown populations were generated. Expression analyses in H9c2 myoblasts demonstrated significant Rbm5 messenger RNA (mRNA) inhibition but, surprisingly, no effect on RBM5 protein levels. Expression of the Rbm5 paralogue Rbm10 was examined in order to (a) ensure no off-target knockdown effect, and (b) investigate any possible compensatory effects. RBM10 protein levels were found to be elevated, in both the clonal and transiently transfected populations. These results suggest that myoblast RBM5 expression is regulated by a process that includes RNA sequestration and/or controlled translation, and that (a) RBM5 function is compensated for by RBM10, and/or (b) RBM5 regulates RBM10 expression. We have developed a model to describe our findings, and suggest further experiments for testing its validity. Since upregulation of Rbm10 might compensate for downregulated Rbm5, and consequently might mask any potential knockdown effect, it could lead to incorrect conclusions regarding the importance of Rbm5 for differentiation. It is therefore imperative to determine how both RBM5 and RBM10 protein expression is regulated.

Vimentin-mediated regulation of cell motility through modulation of beta4 integrin protein levels in oral tumor derived cells

Vimentin expression correlates well with migratory and invasive potential of the carcinoma cells. The molecular mechanism by which vimentin regulates cell motility is not yet clear. Here, we addressed this issue by depleting vimentin in oral squamous cell carcinoma derived cell line. Vimentin knockdown cells showed enhanced adhesion and spreading to laminin-5. However, we found that they were less invasive as compared to the vector control cells. In addition, signaling associated with adhesion behavior of the cell was increased in vimentin knockdown clones. These findings suggest that the normal function of β4 integrin as mechanical adhesive device is enhanced upon vimentin downregulation. As a proof of principle, the compromised invasive potential of vimentin depleted cells could be rescued upon blocking with β4 integrin adhesion-blocking (ASC-8) antibody or downregulation of β4 integrin in vimentin knockdown background. Interestingly, plectin which associates with α6β4 integrin in the hemidesmosomes, was also found to be upregulated in vimentin knockdown clones. Furthermore, experiments on lysosome and proteasome inhibition revealed that perhaps vimentin regulates the turnover of β4 integrin and plectin. Moreover, an inverse association was observed between vimentin expression and β4 integrin in oral squamous cell carcinoma (OSCC). Collectively, our results show a novel role of vimentin in modulating cell motility by destabilizing β4 integrin-mediated adhesive interactions. Further, vimentin-β4 integrin together may prove to be useful markers for prognostication of human oral cancer.

GENO-11PERIOSTIN MAINTAINS A MESENCHYMAL PROGRAM IN GLIOBLASTOMA STEM CELLS

Our radiogenomic screen identified Periostin (POSTN) to be highly expressed in glioblastoma (GBM) with high edema/FLAIR. Subsequently, POSTN is implicated in glioblastoma invasion and recruitment of tumor associated macrophages. However, its impact on glioblastoma stem cells (GSC) biology/properties is largely unknown. Here GSC lines with high endogenous POSTN expression levels were used to generate inducible shRNA mediated loss of function (LOF) clones. GSCs with low/undetectable levels of POSTN expression were used to generate Gain of function (GOF) clones. In vitro tests were performed to assess the impact of POSTN LOF and GOF on GSC properties such as self-renewal, BrdU label retention, differentiation potential etc. We show that GSCs with POSTN LOF acquired an increased stem like state with unaltered proliferation rate. When these clones were implanted at orthotopic locations in mouse, POSTN LOF was associated with increased survival of mice. Cells of human origin were isolated from tumors using human specific HLA Class I ABC antibody by FACS. Total RNA from these sorted cells (without cell-culture) were used for expression profile analysis by HTA 2.0 array from affymetrix. Transcriptome analyses of POSTN LOF cells show inhibition of multiple mesenchymal associated networks such as CEBPB (a master regulator of mesenchymal program in GBM), PRDM1, TGFB1, FGF2, WNT3A, HIF1A etc. with concomitant activation of MYCN network, indicating a mesenchymal to proneural switch. We have generated POSTN and CEBPB dual knockdown clones and are currently investigating the molecular link between these two key mesenchymal genes in GSC context. Our results show that POSTN maintains a mesenchymal program in GSC by coopting with intracellular factors such as CEBPB. Therefore, neutralizing extracellular POSTN will have far reaching impact in terms of therapeutics where while blocking GBM invasion and it may simultaneously block a mesenchymal program in stem cell niche in GBM.

The role of the CCDC26 long noncoding RNA as a tumor suppressor

CCDC26 on chromosome 8q24 is considered to encode a long intergenic noncoding RNA because the short open reading frame within the mRNA transcribed from this gene is not conserved in any other species. Genome-wide analysis has revealed association of CCDC26 with certain tumors, for instance low-level glioma. Moreover, 1.5- to 2-fold amplifications of the whole or part of the CCDC26 genetic locus have been observed in pediatric acute myeloid leukemia patients. The CCDC26 gene is amplified in the HL-60 acute myeloid leukemia cell line, in which double minute chromosomes—abnormal tiny chromosomes—harbor the CCDC26 gene. We examined the function of CCDC26 by gene knock-down (KD) using short hairpin RNAs in K562 human myeloid leukemia cells. In four stable KD clones, CCDC26 expression was suppressed to 1% of its normal level by transcriptional gene suppression, not post-transcriptional suppression. The growth rates of these KD clones were reduced compared with those of control cells in media containing high serum concentrations. In contrast, in media containing much lower serum concentrations, the KD clones exhibited significantly higher growth rates than controls, and increased survival after serum withdrawal. Enhanced expression of a receptor tyrosine kinase, KIT , was detected in the KD clones, and treatment with ISCK03, a KIT inhibitor, eliminated their increased survival in the absence of serum. Therefore, CCDC26 seems to control myeloid leukemia cell growth through regulation of KIT expression. These observations suggest that CCDC26 is a tumor-suppressive long noncoding RNA because it suppresses the KIT oncogene that supports survival of cancer cells in the stem cell state.

Cysteine-rich secretory protein 3 plays a role in prostate cancer cell invasion and affects expression of PSA and ANXA1.

Cysteine-rich secretory protein 3 (CRISP-3) is upregulated in prostate cancer as compared to the normal prostate tissue. Higher expression of CRISP-3 has been linked to poor prognosis and hence it has been thought to act as a prognostic marker for prostate cancer. It is proposed to have a role in innate immunity but its role in prostate cancer is still unknown. In order to understand its function, its expression was stably knocked down in LNCaP cells. CRISP-3 knockdown did not affect cell viability but resulted in reduced invasiveness. Global gene expression changes upon CRISP-3 knockdown were identified by microarray analysis. Microarray data were quantitatively validated by evaluating the expression of seven candidate genes in three independent stable clones. Functional annotation of the differentially expressed genes identified cell adhesion, cell motility, and ion transport to be affected among other biological processes. Prostate-specific antigen (PSA, also known as Kallikrein 3) was the top most downregulated gene whose expression was also validated at protein level. Interestingly, expression of Annexin A1 (ANXA1), a known anti-inflammatory protein, was upregulated upon CRISP-3 knockdown. Re-introduction of CRISP-3 into the knockdown clone reversed the effect on invasiveness and also led to increased PSA expression. These results suggest that overexpression of CRISP-3 in prostate tumor may maintain higher PSA expression and lower ANXA1 expression. Our data also indicate that poor prognosis associated with higher CRISP-3 expression could be due to its role in cell invasion.

Abstract 4483: Impact of loss of folate receptor alpha on antitumor effects of novel 6-substituted pyrrolo[2,3-d]pyrimidine antifolates with substrate activities for both folate receptors and the proton-coupled folate transporter

Abstract Major folate uptake mechanisms include the reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT), and folate receptors α and β. Antifolates such as methotrexate or pemetrexed are excellent substrates for human RFC (hRFC) and variously for human PCFT (hPCFT) and human folate receptor α (hFRα). Recent attention has focused on developing tumor-targeted folate-based therapies, reflecting tumor-selective uptake by hFRα and/or hPCFT. Novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl regioisomers AGF94 (2′,5′) and AGF154 (2′,4′) were synthesized and characterized as potent inhibitors of cell proliferation of engineered Chinese hamster ovary (CHO) cells expressing hPCFT or hFRα, but not for CHO cells expressing hRFC. In 41 ovarian cancer specimens from patients, hFRα, hPCFT and hRFC were all expressed. FRα transcript levels increased with stage of disease, although hPCFT levels were constant. IGROV1 is a clinically relevant ovarian cancer cell line that expresses both hFRα and hPCFT. IGROV1 cells were highly sensitive to both AGF94 and AGF154, as reflected in inhibition of cell proliferation in vitro. Growth inhibition by AGF94 or AGF154 in IGROV1 cells was partially reversed by excess folic acid, likely reflecting drug uptake by hFRα, as well as by hPCFT. To evaluate the impact of loss of hFRα on AGF94 and AGF154 in vitro inhibition of IGROV1 cell proliferation, FRα was knocked down (>95%) by lentivirus shRNAs. Clonal isolates were generated (n = 17) and profiled by RT-PCR for expression of the folate transporters and key enzymes likely to impact antifolate sensitivities (e.g., folylpolyglutamyl synthetase, β-glycinamide ribonucleotide formyltransferase), relative to a non-targeted control (NTC) cells. Gene expression profiles were similar for IGROV1 hFRα knockdown (KD) clones and NTC cells. Two representative hFRα KD clones were assayed for inhibition of cell proliferation by AGF94 and AGF154, with IC50s for IGROV1 KD clones of 26±1.8 and 26±9 nM for AGF94, and 65±1.2 and 81±2.4 nM for AGF154. For comparison, IC50s in the IGROV1 NTC cells expressing both hFRα and hPCFT were 0.6±0.1 nM for AGF94 and 2.2±0.8 nM for AGF154. In contrast, the hFRα-specific antifolate, AGF102 showed IC50s of > 1000 nM and ∼2.5 nM for KD and NTC cells. The preservation of substantial inhibitory activity for AGF94 and AGF154, accompanying the nearly complete loss of FRα expression in the KD clones is likely due to the sustained uptake of these novel antifolates by hPCFT. For AGF154, this was confirmed by direct uptake assays with [3H]-AGF154. Our results suggest that 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates with substantial substrate activities for hFRα and hPCFT may offer significant advantages in treating tumors that express both these transport systems. Citation Format: Zhanjun Hou, Steve Orr, Christina George, Adrianne Wallace, Larry H. Matherly, Lei Wang, Si Yang, Aleem Gandjee. Impact of loss of folate receptor alpha on antitumor effects of novel 6-substituted pyrrolo[2,3-d]pyrimidine antifolates with substrate activities for both folate receptors and the proton-coupled folate transporter. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4483. doi:10.1158/1538-7445.AM2015-4483

Abstract 1251: Expression of NADPH oxidase 5 (Nox5) modulates cellular morphology, proliferation and invasiveness of human melanoma UACC-257 cells

Abstract NADPH oxidase 5 (Nox5), one of the seven members of the NADPH oxidase gene family that generate reactive oxygen species (ROS), has been implicated in physiological and pathophysiological processes. Recently, we have reported substantial overexpression of Nox5 in several human cancers including those of prostate, ovary and melanomas; expression in most non-malignant tissues was weak to modest. Despite being upregulated in many human cancers and implicated in promoting cell proliferation, the molecular mechanisms regulated by Nox5 are poorly understood. In this study, the functional significance of Nox5 was assessed in human UACC-257 melanoma cells by generating stable Nox5-overexpressing clones and knockdown clones. Overexpression of Nox5 in UACC-257 cells resulted in enhanced cell growth, increased BrdU positive cells and decreased phosphatase activity. Additionally, these cells had increased normoxic Hif-1α expression and decreased p27Kip1 expression. Conversely, knockdown of endogenous Nox5 in UACC-257 cells resulted in decreased cell growth, decreased BrdU positive cells, decreased Hif-1α expression and increased p27Kip1 expression. Cadherin switch, loss of E-cadherin expression and upregulation of N-cadherin was observed in Nox5 overexpressing cells indicative of an invasive phenotype; conversely, an upregulation of E-cadherin expression in Nox5 knockdown cells was noted. Cell invasion assay through matrigel-coated membranes also demonstrated enhanced invasion by Nox5 overexpressing cells. Additionally, 3D cultures of Nox5 overexpressing cells exhibit an amoeboid morphology compared to that of the Nox5 knockdown cells that were mesenchymal, suggestive of an amoeboid - mesenchymal (AMT) transition. Strikingly, cells that overexpress Nox5 were able to proliferate in serum-free conditions for over a month. In summary, our findings suggest that Nox5 expression in human melanoma UACC-257 cells could contribute to cell proliferation, invasion, amoeboid morphology, and the ability to grow in the absence of serum or growth factors in part due to the generation of high local concentrations of extracellular ROS that modulate multiple signaling networks that regulate Hif-1α, p27Kip1, and the cadherin switch. Citation Format: Smitha Antony, Yongzhong Wu, Guojian Jiang, Jennifer L. Meitzler, Han Liu, Agnes Juhasz, Jiamo Lu, Miriam R. Anver, Krishnendu K. Roy, James H. Doroshow. Expression of NADPH oxidase 5 (Nox5) modulates cellular morphology, proliferation and invasiveness of human melanoma UACC-257 cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1251. doi:10.1158/1538-7445.AM2015-1251

Abstract 1483: Lipid oxidation via CPT1 as a target for prostate cancer imaging and therapy

Abstract Introduction: Prostate cancer (PCa) is the most common cancer in males and is currently treated medically with androgen deprivation therapy. However, resistance to androgen receptor (AR) targeted therapy develops in almost all patients over time. Simultaneously targeting multiples pathways may prevent the development of this resistance. Recent studies suggest PCa relies on lipid fuel over glycolysis. Blocking the ability of PCa to use lipids inhibits its growth and leads to apoptosis but enhances glucose uptake initially, potentially enhancing glucose-based FDG-PET (18F-dexyglucose-Positron Emission Tomography) imaging of PCa tumors. Experimental approach: To address the role of lipid metabolism in PCa we have used etomoxir, a clinically utilized drug that blocks lipid oxidation by inhibiting carnitine palmitoyltransferase (CPT1) in the mitochondria. Enzalutamide is a clinically available anti-androgen, which was used by itself or in combination with etomoxir. The gene/drug interaction was studied in LNCaP cells with decreased expression of CPT1A (via shRNA). PET-FDG of mouse xenografts were used to evaluated the glycolytic switch induced by systemic etomoxir treatment in 24 hours and the therapeutic effect over time. Results: Treatment with etomoxir alone significantly decreased cell viability and AR content, including ARv7 variant. Combinatorial treatment with enzalutamide synergistically enhanced this effect on viability. CPT1A Knockdown clones were also more sensitive to enzalutamide (2 fold, p<0.001) compared to control clones, and this effect was associated with reduced AR expression. Systemic treatment with etomoxir alone in nude mice resulted in decreased xenograft growth over 21 days, underscoring the therapeutic potential of blocking lipid catabolism to decrease PCa tumor growth. Interestingly, FDG uptake by VCaP xenografts was increased with systemic etomoxir in 24 hours (∼1.5 fold, P<0.05), enhancing the visualization of the tumors in the PET scans. FDG uptake in the mouse non-cancerous prostate tissue was negligible with etomoxir. Conclusions: Safely blocking lipid oxidation in PCa results in decreased viability with temporarily increased glucose uptake, due to a flare of glucose uptake to compensate for the fat oxidation blockade. Validation in human PCa patients with localized disease is needed to confirm these preclinical studies. Additionally, the decreased viability over time suggests that lipid metabolism is needed to maintain AR expression and combination of fat burning inhibitors and enzalutamide may offer novel approach to anti-AR resistance in PCa. Citation Format: Isabel R. Schlaepfer, Maren Salzmann-Sullivan, Lih-Jen Su, L.Michael Glode, Thomas Flaig. Lipid oxidation via CPT1 as a target for prostate cancer imaging and therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1483. doi:10.1158/1538-7445.AM2015-1483

Abstract P6-12-05: Inducible suppression of insulin receptor substrate I inhibits IGF-I/insulin/estradiol dependent cell growth in MCF-7L breast cancer cells

Abstract Insulin receptor substrate (IRS) proteins are adaptor proteins downstream of insulin-like growth factor (IGF) and insulin receptors. IRS proteins play a role in cancer biology, with IRS1 function linked to mitogenesis and survival and IRS2 associated with metastasis. Moreover, this family of adaptor proteins is also involved in the signal transduction of many other transmembrane receptors. Therefore IRS proteins could be potential therapeutic targets for cancer therapy. To test the function of IRS-1, we created a doxycycline inducible IRS1 shRNA and stably transfected MCF-7L breast cancer cells. The level of IRS1 protein knockdown varied within different clones from 50 to 95% reduction compared to non-induced cells. IRS2 mRNA and protein levels were not significantly affected. We chose a high (3G5) and intermediate (3A7) IRS1 knockdown clones for further study. Doxycycline treated 3G5 and 3A7, showed partial inhibition of IGF-I, IGF-II and insulin stimulated phosphorylation of IRS1(Tyr1222) and AKT. pErk1,2 were slightly increased and pIGF-IR was unchanged compared to doxycycline non-treated cells. IRS-2 tyrosine phosphorylation was stimulated when IRS-1 was suppressed and may account for the persistent downstream cell signaling. Anti IGFIR antibody dalotuzumab (20µg/ml) effectively inhibited cell signaling stimulated by IGF-I, IGF-II and insulin in both doxycycline treated and non-treated cells. There was no obvious difference in cell signaling pattern between plus and minus doxycycline treated cells within 24 hour time course. In monolayer cell growth assays, IGF-I, IGF-II, insulin and estradiol growth stimulation were significantly inhibited when IRS-1 was suppressed in 3G5 cells. In 3A7 cells, which have a lower level of IRS-1 suppression, growth stimulation was moderately inhibited. In anchorage independent growth assays stimulated by IGF-I, colony number and size were inhibited by doxycycline-induced IRS-1 suppression with a reduction of 80% in 3G5 cells and 50% reduction in 3A7 cells. These results show that growth of MCF-7L cells are dependent on IRS-1 expression. Reduced IRS1 can hinder cancer cell growth and tumorigenicity even when signaling was not impaired potentially due to compensation by other adaptor proteins. We conclude that suppression of IRS-1 may inhibit several growth regulatory pathways in breast cancer and could serve as a potential drug target. Citation Format: Xihong Zhang, Sidhant Varma, Douglas Yee. Inducible suppression of insulin receptor substrate I inhibits IGF-I/insulin/estradiol dependent cell growth in MCF-7L breast cancer cells [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-12-05.

Long noncoding RNA, CCDC26, controls myeloid leukemia cell growth through regulation of KIT expression.

BackgroundAccumulating evidence suggests that some long noncoding RNAs (lncRNAs) are involved in certain diseases, such as cancer. The lncRNA, CCDC26, is related to childhood acute myeloid leukemia (AML) because its copy number is altered in AML patients.ResultsWe found that CCDC26 transcripts were abundant in the nuclear fraction of K562 human myeloid leukemia cells. To examine the function of CCDC26, gene knockdown (KD) was performed using short hairpin RNAs (shRNAs), and four KD clones, in which CCDC26 expression was suppressed to 1% of its normal level, were isolated. This down-regulation included suppression of CCDC26 intron-containing transcripts (the CCDC26 precursor mRNA), indicating that transcriptional gene suppression (TGS), not post-transcriptional suppression, was occurring. The shRNA targeting one of the two CCDC26 splice variants also suppressed the other splice variant, which is further evidence for TGS. Growth rates of KD clones were reduced compared with non-KD control cells in media containing normal or high serum concentrations. In contrast, enhanced growth rates in media containing much lower serum concentrations and increased survival periods after serum withdrawal were observed for KD clones. DNA microarray and quantitative polymerase chain reaction screening for differentially expressed genes between KD clones and non-KD control cells revealed significant up-regulation of the tyrosine kinase receptor, KIT, hyperactive mutations of which are often found in AML. Treatment of KD clones with ISCK03, a KIT-specific inhibitor, eliminated the increased survival of KD clones in the absence of serum.ConclusionsWe suggest that CCDC26 controls growth of myeloid leukemia cells through regulation of KIT expression. A KIT inhibitor might be an effective treatment against the forms of AML in which CCDC26 is altered.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0364-7) contains supplementary material, which is available to authorized users.

MMP7 is required to mediate cell invasion and tumor formation upon Plakophilin3 loss.

Plakophilin3 (PKP3) loss results in increased transformation in multiple cell lines in vitro and increased tumor formation in vivo. A microarray analysis performed in the PKP3 knockdown clones, identified an inflammation associated gene signature in cell lines derived from stratified epithelia as opposed to cell lines derived from simple epithelia. However, in contrast to the inflammation associated gene signature, the expression of MMP7 was increased upon PKP3 knockdown in all the cell lines tested. Using vector driven RNA interference, it was demonstrated that MMP7 was required for in-vitro cell migration and invasion and tumor formation in vivo. The increase in MMP7 levels was due to the increase in levels of the Phosphatase of Regenerating Liver3 (PRL3), which is observed upon PKP3 loss. The results suggest that MMP7 over-expression may be one of the mechanisms by which PKP3 loss leads to increased cell invasion and tumor formation.