Independent Cell Lines Research Articles

Circular RNAs are down-regulated in KRAS mutant colon cancer cells and can be transferred to exosomes.

Recent studies have shown that circular RNAs (circRNAs) are abundant, widely expressed in mammals, and can display cell-type specific expression. However, how production of circRNAs is regulated and their precise biological function remains largely unknown. To study how circRNAs might be regulated during colorectal cancer progression, we used three isogenic colon cancer cell lines that differ only in KRAS mutation status. Cellular RNAs from the parental DLD-1 cells that contain both wild-type and G13D mutant KRAS alleles and isogenically-matched derivative cell lines, DKO-1 (mutant KRAS allele only) and DKs-8 (wild-type KRAS allele only) were analyzed using RNA-Seq. We developed a bioinformatics pipeline to identify and evaluate circRNA candidates from RNA-Seq data. Hundreds of high-quality circRNA candidates were identified in each cell line. Remarkably, circRNAs were significantly down-regulated at a global level in DLD-1 and DKO-1 cells compared to DKs-8 cells, indicating a widespread effect of mutant KRAS on circRNA abundance. This finding was confirmed in two independent colon cancer cell lines HCT116 (KRAS mutant) and HKe3 (KRAS WT). In all three cell lines, circRNAs were also found in secreted extracellular-vesicles, and circRNAs were more abundant in exosomes than cells. Our results suggest that circRNAs may serve as promising cancer biomarkers.

Effect of lactate and pH on mouse pluripotent stem cells: Importance of media analysis

Pluripotent stem cells have generated a great deal of excitement concerning their use for clinical therapy and regenerative medicine. Similar to other mammalian cells, the growth of these cells is dependent on various culture conditions. The presence of lactic acid as a metabolic by-product has been reported to be detrimental for mammalian cells in most cases. However, the use of lactate as a source of energy has also been reported for certain cell types. The current study was carried out to examine the effect of media lactate concentration and extracellular pH change, two important parameters that occur due to lactic acid production, on mouse pluripotent stem cells, both embryonic and induced pluripotent stem cells. We examined both feeder dependent and feeder independent embryonic stem cell lines for a comprehensive observation. It was noted that increase or decrease in pH affected cell proliferation, viability and pluripotency of all three cell lines. The effect of lactate was less obvious. Supplementation with lactate decreased cell proliferation and cell number. However, from the data obtained, we hypothesize that the surviving cells were able to adapt to the change in environment and utilize lactate as an energy source. There was no significant effect of lactate on the pluripotency of the cells.

A Viral Immunity Chromosome in the Marine Picoeukaryote, Ostreococcus tauri.

Micro-algae of the genus Ostreococcus and related species of the order Mamiellales are globally distributed in the photic zone of world's oceans where they contribute to fixation of atmospheric carbon and production of oxygen, besides providing a primary source of nutrition in the food web. Their tiny size, simple cells, ease of culture, compact genomes and susceptibility to the most abundant large DNA viruses in the sea render them attractive as models for integrative marine biology. In culture, spontaneous resistance to viruses occurs frequently. Here, we show that virus-producing resistant cell lines arise in many independent cell lines during lytic infections, but over two years, more and more of these lines stop producing viruses. We observed sweeping over-expression of all genes in more than half of chromosome 19 in resistant lines, and karyotypic analyses showed physical rearrangements of this chromosome. Chromosome 19 has an unusual genetic structure whose equivalent is found in all of the sequenced genomes in this ecologically important group of green algae.

Combined targeting of SET and tyrosine kinases provides an effective therapeutic approach in human T-cell acute lymphoblastic leukemia.

Recent evidence suggests that inhibition of protein phosphatase 2A (PP2A) tumor suppressor activity via the SET oncoprotein contributes to the pathogenesis of various cancers. Here we demonstrate that both SET and c-MYC expression are frequently elevated in T-ALL cell lines and primary samples compared to healthy T cells. Treatment of T-ALL cells with the SET antagonist OP449 restored the activity of PP2A and reduced SET interaction with the PP2A catalytic subunit, resulting in a decrease in cell viability and c-MYC expression in a dose-dependent manner. Since a tight balance between phosphatases and kinases is required for the growth of both normal and malignant cells, we sought to identify a kinase inhibitor that would synergize with SET antagonism. We tested various T-ALL cell lines against a small-molecule inhibitor screen of 66 compounds targeting two-thirds of the tyrosine kinome and found that combined treatment of T-ALL cells with dovitinib, an orally active multi-targeted small-molecule receptor tyrosine kinase inhibitor, and OP449 synergistically reduced the viability of all tested T-ALL cell lines. Mechanistically, combined treatment with OP449 and dovitinib decreased total and phospho c-MYC levels and reduced ERK1/2, AKT, and p70S6 kinase activity in both NOTCH-dependent and independent T-ALL cell lines. Overall, these results suggest that combined targeting of tyrosine kinases and activation of serine/threonine phosphatases may offer novel therapeutic strategies for the treatment of T-ALL.

Rapid induction of GFP expression by the nitrate reductase promoter in the diatom Phaeodactylum tricornutum.

An essential prerequisite for a controlled transgene expression is the choice of a suitable promoter. In the model diatom Phaeodactylum tricornutum, the most commonly used promoters for trans-gene expression are the light dependent lhcf1 promoters (derived from two endogenous genes encoding fucoxanthin chlorophyll a/c binding proteins) and the nitrate dependent nr promoter (derived from the endogenous nitrate reductase gene). In this study, we investigated the time dependent expression of the green fluorescent protein (GFP) reporter under control of the nitrate reductase promoter in independently genetically transformed P. tricornutum cell lines following induction of expression by change of the nitrogen source in the medium via flow cytometry, microscopy and western blotting. In all investigated cell lines, GFP fluorescence started to increase 1 h after change of the medium, the fastest increase rates were observed between 2 and 3 h. Fluorescence continued to increase slightly for up to 7 h even after transfer of the cells to ammonium medium. The subsequent decrease of GFP fluorescence was much slower than the increase, probably due to the stability of GFP. The investigation of several cell lines transformed with nr based constructs revealed that, also in the absence of nitrate, the promoter may show residual activity. Furthermore, we observed a strong variation of gene expression between independent cell lines, emphasising the importance of a thorough characterisation of genetically modified cell lines and their individual expression patterns.

Synthesis of novel C2-symmetric testosterone dimers and evaluation of antiproliferative activity on androgen-dependent and -independent prostate cancer cell lines

A series of 7α-linked testosterone dimers were made and tested for biological activity on both androgen-dependent (LNCaP) and androgen-independent (DU-145 and PC3) prostate cancer cell lines. The synthesis proceeds through the formation of a trans-4-(17β-acetoxy-4-androsten-3-one-7α-yl)-but-2-enoic acid 4-hydroxy-alkyl ester intermediate of various length (7a–d) followed by the final dimerization step. The dimers showed interesting biological activity in comparison to the ω-hydroxyalkyl ester intermediates 7a–d. The most active dimer 8a (n=1) showed IC50 of 3.8, 1.4 and 1.8μM, respectively on LNCaP, DU-145 and PC3 cancer cell lines. On these cell lines, this dimer is about 12, 70 and 47 times more powerful than cyproterone acetate (CPA) the reference antiandrogen. Furthermore, dimers 8b–d (n=2, 3, 4) were less active than 8a but showed selective activity on androgen-dependent LNCaP prostate cancer cells. This indicates possible application for the treatment of androgen-dependent prostate cancer.

Mouse fibroblasts null for the long isoform of β1,4-galactosyltransferase-I show defective cell-matrix interactions

β1,4 Galactosyltransferase-I (GalT-I) is expressed as two nearly identical polypeptides that differ only in the length of their cytoplasmic domains. The longer isoform has been implicated as a cell surface receptor for extracellular glycoside ligands, such as laminin. To more stringently test the function of the long GalT-I isoform during cell interactions with laminin, we created multiple independent fibroblastic cell lines that fail to express the long isoform, but which express the short GalT-I isoform normally and appear to have normal intracellular galactosylation. Cells devoid of the long GalT-I isoform are unable to adhere and spread on laminin substrates as well as control cells, but retain near normal interactions with fibronectin, which do not rely upon surface GalT-I function. The loss of the long GalT-I isoform also leads to a loss of actin stress fibers, focal adhesions and rac GTPase activation.

The functional status of DNA repair pathways determines the sensitization effect to cisplatin in non-small cell lung cancer cells.

Cisplatin can cause a variety of DNA crosslink lesions including intra-strand and inter-strand crosslinks (ICLs), which are associated with the sensitivity of cancer cells to cisplatin. Here, we aimed to assess the contribution of the Fanconi anemia (FA), homologous recombination (HR) and nucleotide excision repair (NER) pathways to cisplatin resistance in non-small cell lung cancer (NSCLC)-derived cells. The expression of FA, HR and NER pathway-associated genes was assessed by RT-qPCR and Western blotting. siRNAs were used to knock down the expression of these genes. CCK-8 and flow cytometry assays were used to assess the viability and apoptotic rate of NSCLC-derived cells, respectively. Immunofluorescence and alkaline comet assays were used to assess the repair of ICLs. We found that acquired cisplatin-resistant NSCLC-derived A549/DR cells exhibited markedly enhanced FA and HR repair pathway capacities compared to its parental A549 cells and another independent NSCLC-derived cell line, Calu-1, which possesses a moderate innate resistance to cisplatin. siRNA-mediated silencing of the FA-associated genes FANCL and RAD18 and the HR-associated genes BRCA1 and BRCA2 significantly potentiated the sensitivity of A549/DR cells to cisplatin compared to A549 and Calu-1 cells, suggesting that the acquired cisplatin resistance in A549/DR cells may be attributed to enhanced FA and HR pathway capacities responsible for ICL repair. Although we found that expression knockdown of the NER-associated genes XPA and ERCC1 sensitized the three NSCLC-derived cell lines to cisplatin, the sensitization effect was more significant in Calu-1 cells than in A549 and A549/DR cells, implying that the innate cisplatin resistance in Calu-1 cells may result from an increased NER activity. Our results indicate that the functional status of DNA repair pathways determine the sensitivity of NSCLC cells to cisplatin. Direct targeting of the pathway that is involved in cisplatin resistance may be an effective strategy to surmount cisplatin resistance in NSCLC.

Abstract 1360: Genistein-AR antagonist conjugate potently induce growth arrest in both androgen dependent and independent prostate cancer cell lines

Abstract The ability of prostate cancer to transition from androgen dependent- to androgen independent state (castration resistant), during androgen deprivation therapy, requires that new chemotherapeutic agents be introduced that will retain activity in the two prostate cancer types. Though described as androgen independent, castration resistant prostate cancer (CRPC) still relies heavily on androgen receptor (AR) signaling for survival. Enzalutamide, an AR antagonist, is probably that most effective drug used in androgen deprivation therapy for CRPC and AR dependent prostate cancer. Despite its clinical success, there is the fear of resistant to enzalutamide due to mutation in the ligand binding domain of AR. This poses a challenge that requires developing new agents devoid of resistance, in the event of mutation. One of the approaches being proposed is to develop new agents that can selectively induce AR degradation, an approach that was successfully used to develop the selective estrogen receptor degrader, fulvestrant. In our study, we incorporate genistein, a soy isoflavone known to perturb multiple biochemical pathways relevant for cancer survival, into an AR binding template to give AG-1-33. AG-1-33 was evaluated for growth inhibitory activity in LNCaP (AR dependent) and DU-145 (AR independent) prostate cancer cell lines. This compound retained its AR antagonist activity and also showed low micromolar anti-proliferation activity in the two prostate cancer cell lines. We also evaluated the effect of AG-1-33 on AR expression, as well as its ability to act as a selective AR degrader in LNCaP. Citation Format: Idris O. Raji, Alex George, Omer Kucuk, Adegboyega K. Oyelere. Genistein-AR antagonist conjugate potently induce growth arrest in both androgen dependent and independent prostate cancer cell lines. [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 1360.

Abstract 1134: Association of ZC3H8 with nuclear bodies and its role in promoting tumor cell behavior in vitro and in vivo

Abstract The Zinc Finger CCCH-Type Containing 8 (zc3h8 or Fliz1) gene is overexpressed in many mouse and human breast cancer cell lines that we examined, although the exact function of this gene and the ZC3H8 protein are largely unknown. To characterize ZC3H8 protein, we localized it to distinct sub-nuclear domains that also contain markers for promyelocytic leukemia (PML) nuclear bodies and Cajal bodies. Furthermore, when cells are treated with a casein kinase-2 (CK2) inhibitor, ZC3H8 mislocalizes out of the nuclear bodies and diffuses into the nucleoplasm. CK2 is a known activator of transcription and a potential drug target. ZC3H8 contains a putative CK2 phosphorylation site at position T32, and may be regulated by this kinase. Based on this localization and other preliminary data, we hypothesize that ZC3H8 has a role in transcriptional regulation or RNA modification at these nuclear bodies. This hypothesis is supported by published studies that have found a role for ZC3H8 in the Little Elongation Complex (LEC), which transcribes snRNA involved in RNA splicing in nuclear bodies. Since many tumors and cancer cell lines overexpress zc3h8, we used stably expressed shRNA to knock down expression in two independent mouse breast cancer cell lines for in vitro and in vivo studies. Cells with reduced zc3h8 expression have decreased rates of proliferation and migration compared to overexpressing cells when tested using MTS and wound healing assays. Decreased rates of migration in wound healing assays are reversed by overexpression of the known migration-associated gene MEMO1. Additionally, cells with reduced expression of zc3h8 failed to form colonies in soft agar or pass through an extracellular matrix coating a transwell chamber- a phenotype that can be rescued using recombinant human zc3h8. Conversely, untransformed mouse mammary COMMA-1D cells gain the ability to form colonies in soft agar when transfected to stably overexpress zc3h8. Our in vitro studies suggest that zc3h8 expression has a role in tumorigenesis. These observations have been supported with in vivo studies in mice. We implanted cells knocked down for zc3h8 and control cells into the mammary glands of syngeneic female mice. Mice injected with cells that have reduced expression of zc3h8 form substantially smaller tumors by weight compared to control. When taken together, these data show that zc3h8 has a role in the formation and progression of cancer through its association with nuclear bodies involved in transcriptional regulation and RNA modification. Citation Format: John A. Schmidt, Keith G. Danielson, Jani L. Swiatek, Emily R. Duffner, Janice E. Knepper. Association of ZC3H8 with nuclear bodies and its role in promoting tumor cell behavior in vitro and in vivo. [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 1134.

Abstract 2907: Eugenol potentiates the effect of cisplatin on cancer stem-like cells through targeting the NF-κB pathway

Abstract Triple-negative subtype of breast cancer (TNBC) contains exclusively tumorigenic and often endowed with self-renewing and resistance to cytotoxic chemotherapy, such as, paclitaxel resulting in relative increases in CSCs phenotype. Signaling through NF-kB may be essential for the CSCs self-renewal and could present potential target for novel treatment in breast cancer patients. We explored the possible involvement of NF-kB pathway that contribute to the functions and maintenance of breast CSCs and targeting this pathway by cisplatin combining with a natural product eugenol both in vitro and in vivo, using estrogen dependent (MCF-7) and independent (MDA-MB-231, MDA-MB-468 and BT-20) cell lines and xenografted both subcutaneous and orthotopic (fat pad) sites in nude mice. Chromatin immunoprecipitation (ChIP) assay was utilized to assess the NF-kB associated DNA binding capacity in promoter region. Surprisingly, neither cisplatin nor eugenol was capable of complete diminishing the CSCs populations. Cisplatin alone administration partially diminished apoptosis, mammospheres formation, aldehyde dehydrogenase 1 (ALDH1) activity, invasion and growth on nude mice. We observed cisplatin-induced activation of NF-kB associated with survival and regrowth of mamospheres. For effective elimination of CSCs phenotype by cisplatin, we cotreated cells with eugenol. Combination of eugenol and cisplatin significantly eradicated cisplatin induced NF-kB DNA binding capability assed by EMSA and ChIP assays, which was associated with abrogated apoptosis, mammospheres formation, ALDH1 activity and invasion. In vivo, combination therapy reduced the tumor size in synergistic manner. This was possibly due to induction of apoptosis, inhibition of proliferation, angiogenesis and downregulation of cisplatin-induced expression of proteins of NF-kB target genes. Although treatment of mammospheres by cisplatin partially induced the CSCs population through interleukin (IL)-6 pathway, however, only coadministration of eugenol and cisplatin reduced the numbers of CSCs to virtually undeletable levels in vitro and in vivo. Our data suggest that eugenol may be well suited to increase targeting of breast CSCs by cisplatin. Citation Format: Syed S. Islam, Al-Sharif Ibtehaj, Sultan Ahlam, Abdelilah Aboussekhra. Eugenol potentiates the effect of cisplatin on cancer stem-like cells through targeting the NF-κB pathway. [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 2907.

Abstract 1340: Dextran-Catechin conjugate: An anticancer nano-modified natural compound targeting copper metabolism in neuroblastoma

Abstract Background: Neuroblastoma is an aggressive childhood cancer that is poorly responsive to therapy. Moreover, survivors experience long term side effects from their treatment highlighting the need for effective and less toxic therapies. Catechin is a natural polyphenol with anti-cancer properties and limited side effects. Low serum stability of Catechin limits its clinical use that we overcame by chemical functionalization with Dextran. However its mechanism of action is unknown. Here, we investigated the mechanism of action of Dextran-Catechin, and its efficacy against neuroblastoma in vitro and in vivo. Methods: Anticancer activity was tested in 4 independent neuroblastoma cell lines using the cell viability assay Alamar Blue and apoptosis was assessed using PARP cleavage. Gene expression was determined using qRT-PCR and protein expression of CTR1 by western blotting. Intracellular copper was measured by spectrophotometric analysis. Fluorescence-lifetime imaging microscopy was used to study NADH/NAD+ ratio to determine the induction of oxidative stress. Cellular levels of the antioxidant GSH was examined using a colorimetric assay. PET imaging studies with Cu64 were performed in a xenograft neuroblastoma model to monitor copper uptake in tumors. In vivo anticancer activity of Dextran-Catechin was assessed in human xenograft and syngeneic models of neuroblastoma. Results: The neuroblastoma cell lines SH-SY5Y, IMR-32, BE(2)C and doxorubicin-resistant BE(2)C-ADR were sensitive to Dextran-Catechin (IC50 9.7 μg/ml, 17.83 μg/ml, 16 μg/ml and 18.2 μg/ml, respectively) at concentrations that were not toxic to non-malignant MRC-5 cells. However, Cisplatin-resistant neuroblastoma cells, IMR-32-CisRes, were 2.5-fold resistant against Dextran-Catechin compared to the parental cells. Copper transporter 1 (CTR1) mediates cisplatin uptake and IMR-32-CisRes exhibited 50% lower expression of CTR1 and lower intracellular copper compared to the IMR-32 cells. In contrast, Dextran-Catechin sensitive neuroblastoma cells had elevated intracellular copper implicating copper in the activity of this conjugate. We demonstrated that Dextran-Catechin reacts with copper generating reactive oxygen species and inducing cancer cell death. Dextran-Catechin treatment caused a decrease of NADH/NAD+ ratio and GSH levels, confirming oxidative stress. PET imaging analysis of the IMR-32-neuroblastoma xenograft model revealed high accumulation of copper in the tumor mass. The high levels of copper were maintained in the tumor mass for up to 48h, while Cu64 was cleared by the other organs. Importantly, we showed that Dextran-Catechin significantly reduced tumor growth in human xenograft and syngeneic models of neuroblastoma with no evidence of side effects. Conclusion: Dextran-Catechin targets copper, inhibits tumour growth, and has therapeutic potential for cancers dependent on copper for their growth. Citation Format: Orazio Vittorio, Miriam Brandl, Giuseppe Cirillo, Kathleen Kimpton, Elizabeth Hinde, Hien T. T. Duong, Cyrille Boyer, Claudia Flemming, Eugene M.H. Yee, Naresh Kumar, Arvind Parmar, Giancarlo Pascali, Arnaud Charil, Michelle Haber, Murray Norris, Maria Kavallaris. Dextran-Catechin conjugate: An anticancer nano-modified natural compound targeting copper metabolism in neuroblastoma. [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 1340.

Abstract 4131: Blocking endogenous TBX2 abrogates prostate cancer bone metastasis through WNT signaling

Abstract Bone metastasis is a crucial turning point in the progression of PCa and is seen in > 90% of the lethal cases. Identifying factors that mediate the metastatic spread and subsequent remodeling of the bone is highly relevant to successful therapeutic intervention in advanced human prostate cancer (PCa). TBX2, a transcription factor that belongs to the T-box family negatively regulates p21 cell cycle inhibitor and is known to play critical roles during embryonic development and cancer progression. Interestingly, a recent report found TBX2 over-expression in the bone metastases from patients with castrate resistant prostate cancer. We found TBX2 over-expression in human prostate cancer specimens and in aggressive androgen independent human PCa cell lines. Further, we found TBX2 over-expression in bone metastases in multiple xenograft mouse models of human PCa progression. Blocking endogenous TBX2 in PC3 and ARCaPM PCa cells through the use of a dominant negative construct resulted in decreased proliferation, colony formation and invasion in vitro. Utilizing various pre-clinical xenograft mouse models, we found that blocking endogenous TBX2 in PCa cells leads to decreased invasion and abrogation of bone and soft tissue metastasis in vivo. Further, utilizing the intra-tibial mouse model of growth in the bone microenvironment, we found that blocking endogenous TBX2 in PCa cells dramatically reduces bone remodeling. Additionally we found that TBX2 acts upstream of the canonical WNT (WNT3A) signaling in PCa and positively regulates its transcription by binding to its promoter. Further, rescue of WNT3A levels in PCa cells in the context of blocking endogenous TBX2 rescues the metastatic ability of the cells in vivo. Taken together, our findings highlight a novel and critical role of TBX2 in PCa progression, metastasis and the subsequent bone remodeling, and that TBX2 function in PCa is mediated through the canonical WNT signaling. Citation Format: Srinivas Nandana, Manisha Tripathi, Peng Duan, Gina Chu, Haiyen E. Zhau, Robert J. Matusik, Leland W.K. Chung. Blocking endogenous TBX2 abrogates prostate cancer bone metastasis through WNT signaling. [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 4131.

Abstract 1584: Musashi-2 (MSI2) drives TGFBR1/SMAD3 dependent partial EMT and supports VEGFR2 expression and metastasis of human and mouse NSCLC cells

Abstract About 221,200 new patients will be diagnosed with lung cancer and ∼158,040 will succumb to this disease in the United States in 2015. Non-small cell lung cancer (NSCLC) has a 17.4% overall 5-year survival, with metastasis contributing to the vast majority of deaths. Analyzing NSCLC tumors spontaneously arising in KrasLA1/+; P53R172HG/+ (KP) mice, we identified Musashi-2 (MSI2) protein, a stem cell-associated factor that is regulates mRNA translation, as upregulated in the metastasis-competent mouse cell lines. Importantly, MSI2 shRNA depletion in either mouse or human NSCLC cells decreased invasion in Matrigel in vitro and decreased metastasis upon orthotopic injection 129Sv immunocompetent mice in vivo. Mechanistically, by both overexpressing Msi2 cDNA in 393p murine NSCLC and shRNA depleting MSI2 in four independent mouse and human NSCLC cell lines, we defined MSI2 as a driver of a partial epithelial-mesenchymal transition (EMT) program in NSCLC cells. In support of EMT, MSI2 increases the expression of the Snail and Slug pro-EMT transcription factors, as well as the mesenchymal protein vimentin (VMN). MSI2 also downregulates expression of the extracellular matrix component fibronectin (FN1) and tight junction proteins Claudin-3, 5 and 7. However, MSI2 also inhibits protein expression of Zeb-1 and Zeb-2, while sustaining expression of E-cadherin (E-Cad), associated with epithelial identity. Moreover, MSI2 represses NOTCH-1 and upregulates VEGFR2 at the mRNA and protein levels. This is of interest, as NOTCH-1 has been shown to regulate VEGFR2 in angiogenic signaling. We found that knockdown of NOTCH-1 in MSI2-depleted mouse and human NSCLC cells rescued the loss of VEGFR2 expression, suggesting MSI2 increases VEGFR2 expression in a NOTCH-1 dependent manner. Additionally, siRNA of VEGFR2 in the highly metastatic NSCLC cell line 344sq significantly decreased Matrigel invasion, but had only a limited effect in 344sq derivative lines with stable depletion of MSI2, and human NSCLC experiments are ongoing. Together, these results indicate a possible role of MSI2/NOTCH-1/VEGFR2 axis in NSCLC, and suggest that MSI2 connects cancer cell stemness, EMT, and angiogenesis in lung cancer metastasis. Citation Format: Alexander Kudinov, Alexander Deneka, Anna Nikonova, Ilya Serebriiskii, Tim N. Beck, Qi Cai, Brian L. Egleston, Emmanuelle Nicolas, Hossein Borghaei, Don Gibbons, Jonathan Kurie, Erica A. Golemis, Yanis Boumber. Musashi-2 (MSI2) drives TGFBR1/SMAD3 dependent partial EMT and supports VEGFR2 expression and metastasis of human and mouse NSCLC cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1584.

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.

Tumorigenic potential is restored during differentiation in fusion-reprogrammed cancer cells.

Detailed understanding of the mechanistic steps underlying tumor initiation and malignant progression is critical for insights of potentially novel therapeutic modalities. Cellular reprogramming is an approach of particular interest because it can provide a means to reset the differentiation state of the cancer cells and to revert these cells to a state of non-malignancy. Here, we investigated the relationship between cellular differentiation and malignant progression by the fusion of four independent mouse cancer cell lines from different tissues, each with differing developmental potentials, to pluripotent mouse embryonic stem (ES) cells. Fusion was accompanied by loss of differentiated properties of the four parental cancer cell lines and concomitant emergence of pluripotency, demonstrating the feasibility to reprogram the malignant and differentiative properties of cancer cells. However, the original malignant and differentiative phenotypes re-emerge upon withdrawal of the fused cells from the embryonic environment in which they were maintained. cDNA array analysis of the malignant hepatoma progression implicated a role for Foxa1, and silencing Foxa1 prevented the re-emergence of malignant and differentiation-associated gene expression. Our findings support the hypothesis that tumor progression results from deregulation of stem cells, and our approach provides a strategy to analyze possible mechanisms in the cancer initiation.

Musashi-2 (MSI2) supports TGF-β signaling and inhibits claudins to promote non-small cell lung cancer (NSCLC) metastasis

Non-small cell lung cancer (NSCLC) has a 5-y survival rate of ∼16%, with most deaths associated with uncontrolled metastasis. We screened for stem cell identity-related genes preferentially expressed in a panel of cell lines with high versus low metastatic potential, derived from NSCLC tumors of Kras(LA1/+);P53(R172HΔG/+) (KP) mice. The Musashi-2 (MSI2) protein, a regulator of mRNA translation, was consistently elevated in metastasis-competent cell lines. MSI2 was overexpressed in 123 human NSCLC tumor specimens versus normal lung, whereas higher expression was associated with disease progression in an independent set of matched normal/primary tumor/lymph node specimens. Depletion of MSI2 in multiple independent metastatic murine and human NSCLC cell lines reduced invasion and metastatic potential, independent of an effect on proliferation. MSI2 depletion significantly induced expression of proteins associated with epithelial identity, including tight junction proteins [claudin 3 (CLDN3), claudin 5 (CLDN5), and claudin 7 (CLDN7)] and down-regulated direct translational targets associated with epithelial-mesenchymal transition, including the TGF-β receptor 1 (TGFβR1), the small mothers against decapentaplegic homolog 3 (SMAD3), and the zinc finger proteins SNAI1 (SNAIL) and SNAI2 (SLUG). Overexpression of TGFβRI reversed the loss of invasion associated with MSI2 depletion, whereas overexpression of CLDN7 inhibited MSI2-dependent invasion. Unexpectedly, MSI2 depletion reduced E-cadherin expression, reflecting a mixed epithelial-mesenchymal phenotype. Based on this work, we propose that MSI2 provides essential support for TGFβR1/SMAD3 signaling and contributes to invasive adenocarcinoma of the lung and may serve as a predictive biomarker of NSCLC aggressiveness.

A Fucus vesiculosus extract inhibits estrogen receptor activation and induces cell death in female cancer cell lines.

BackgroundWe previously reported the anti-estrogenic activity of the brown seaweed, Fucus vesiculosus. The present study aimed to further investigate its anti-estrogenic modes of action and to assess other potentially biologically relevant anti-tumorigenic effects in estrogen receptor (ER)-dependent and -independent female cancer cell lines.MethodsThe CALUX® assay was used to determine the effect of a F. vesiculosus extract (FVE) on activation of the ER. Aromatase enzymatic activity was measured to determine the potential effect of FVE on estradiol (E2) biosynthesis. Transcriptional activity profiling of 248 genes involved in cancer, immunity, hormonal regulation, protein phosphorylation, transcription, metabolism, and cellular structure was conducted using the NanoString nCounter® analysis system in FVE-treated breast, ovarian and endometrial cancer cell lines. The effects of FVE on cell viability, morphology, membrane integrity, mitochondrial toxicity, induction of apoptotic and autophagic markers, and cell signaling were also analyzed.ResultsIn co-treatments with 12.5 pM (EC50) E2, FVE (2 %) reduced ER activation by 50 %, exhibiting potent ER antagonistic effects. FVE inhibited aromatase activity in an in vitro assay (IC50 2.0 %). ER-dependent and -independent cancer cell lines showed significantly decreased viability that correlated with increasing FVE concentrations and altered morphological features suggestive of apoptosis and autophagy. Expression of genes that were significantly altered by FVE (p < 0.05) revealed predominantly apoptotic, autophagic and kinase signaling pathways. FVE also effectively inhibited the phosphorylation of Akt, resulting in reduced mTORC1 activities to stimulate autophagy in cells. Concentration-dependent cleavage of PARP and induction of caspase-3 and -7 activities were observed in MDA-MB-231 cells supporting a role for FVE in the promotion of apoptosis.ConclusionsOur study provides new insights into the anti-estrogenic activity of F. vesiculosus. Moreover, the induction of autophagy and apoptosis on breast, endometrial and ovarian cancer cell lines suggests additional anti-tumorigenic actions of FVE that are independent of ER status in female cancers.Electronic supplementary materialThe online version of this article (doi:10.1186/s12906-016-1129-6) contains supplementary material, which is available to authorized users.

286. Genome Editing of Inducible Cell Lines for Scalable Production of Improved Lentiviral Vectors for Human Gene Therapy

Lentiviral vectors (LVs) represent efficient and versatile vehicles for gene therapy. Current manufacturing of clinical-grade LVs mostly relies on transient transfection of plasmids expressing the multiple vector components. This method is labor and cost intensive and becomes challenging when facing the need of scale-up and standardization. The development of stable LV producer cell lines will greatly facilitate overcoming these hurdles. We have generated an inducible LV packaging cell line, carrying the genes encoding for third-generation vector components stably integrated in the genome under the control of tetracycline-regulated promoters. These LV packaging cells are stable in culture even after single-cell cloning and can be scaled up to large volumes. In order to minimize the immunogenicity of LVs for in vivo administration, we set out to remove the highly polymorphic class-I major histocompatibility complexes (MHC-I) expressed on LV packaging cells and incorporated in the LV envelope. We performed genetic disruption of the β-2 microglobulin (B2M) gene, a required component for the assembly and trafficking of all MHC-I to the plasma membrane in LV producer cells, exploiting the RNA-guided Cas9 nuclease. The resulting B2M-negative cells were devoid of surface-exposed MHC-I and produced MHC-free LVs. These LVs retain their infectivity on all tested cells in vitro and efficiently transduced the mouse liver upon intravenous administration. Strikingly, the MHC-free LVs showed significantly reduced immunogenicity in a T-cell activation assay performed on human primary T cells co-cultured with syngeneic monocytes exposed to LV, from several (n=7) healthy donors. To reproducibly generate LV-producer cell lines from these cells, we insert the LV genome of interest in the AAVS1 locus, chosen for robust expression, exploiting engineered nucleases and homology-directed repair. By this strategy, we have obtained several independent producer cell lines for LVs that express marker or therapeutic genes and are devoid of plasmid DNA contamination. LVs produced by these cells reproducibly show titer and infectivity within the lower bound range of standard optimized transient transfection, and effectively transduce relevant target cells, such as hematopoietic stem/progenitor cells and T cells ex vivo and the mouse liver in vivo. Overall, we provide evidence that rationally designed targeted genome engineering can be used to improve the yield, quality, safety and sustainability of LV production for clinical use.

Design and evaluation of novel oxadiazole derivatives as potential prostate cancer agents

Various 1,3,4-oxadiazole derivatives have been synthesized and their antiproliferative properties have been studied. The in vitro screening was performed against androgen dependent (LNCaP) and androgen independent (PC-3) prostate cancer cell lines. Most of the compounds showed promising activity. Among them, compounds 2d (IC50=0.22 and 1.3μM) and 2a (IC50=8.34 and 2,5μM) have shown significant activities on PC-3 and LNCaP cell lines respectively. To investigate the mechanism of cell death we performed cell apoptosis staining and cell cycle arrest assay on more sensitive PC-3 cell lines on 2d. The results demonstrated that 2d induced apoptosis and shifted the cells to the sub G0/G1 and S phase. Our study evidently identified the potency of compound 2d as potential anti-prostate cancer agent.