Non-adherent Growth Conditions Research Articles

STEM-05. GLIOBLASTOMA NEUROSPHERES DISPLAY STEMNESS CHARACTERISTICS AND UPREGULATION OF THE VEGF-A PATHWAY

Abstract INTRODUCTION Glioma stem cells (GSC) constitute a subpopulation of tumor cells with stem-cell like properties and are believed to enhance glioblastoma’s ability to grow and confer resistance against chemoradiotherapy. It has been shown that non-adherent three-dimensional (3D) growth conditions facilitate neurosphere formation and enable isolation of GSCs in vitro. This can serve as an invaluable tool to better understand the role of GSCs in glioblastoma pathogenesis. OBJECTIVE We aimed to evaluate differentially expressed genes in non-adherent versus adherent conditions using primary tumor glioblastoma cell lines; and to identify alterations in various signaling pathways that are paramount to their growth and maintenance. Furthermore, we targeted the EMP2-HIF1α axis, a proposed regulator of VEGF expression, to determine its effects on cell viability. METHODS Three primary tumor glioblastoma cell lines were generated and cultured under adherent or non-adherent growth conditions. Sequencing was performed on the Illumina HiSeq3000 system, and gene counts were normalized by TMM. A pair-wise statistical analysis of over 20,000 genes was performed between the two conditions. A p-value < 0.05 was considered statistically significant. RESULTS After 72 hours of growth under 3D conditions, primary glioblastoma cells show an increase in stem-like properties, measured via an upregulation of SOX2 and nestin. RNA sequencing demonstrated a significant increase in expression of the following transcripts in 3D conditions compared to those grown on adherent plates: VEGF-A, EGFR, PDGFR-A, FGFR3, and TGFB2. Treatment with anti-EMP2 monoclonal antibodies significantly reduced cellular proliferation. CONCLUSION Our results demonstrate that neurospheres upregulate growth factor pathways to enable their survival in 3D conditions, suggesting an inherently different genetic landscape between differentiated tumor cells and GSCs. Future studies will investigate the cellular mechanisms responsible for upregulation of these genes; and to ultimately design more effective targeted therapeutic agents.

Abstract 2656: Using Nanog expression to identify breast cancer stem cell populations.

Abstract Many tumors have been demonstrated to contain a subpopulation of cancer stem cells (CSC) whose epigenetic modifications convey resistance to conventional therapies. Furthermore, tumor expression of genes associated with pluripotent embryonic cells, such as Oct-4, Sox-2 and Nanog, often correlates with poor prognosis. We hypothesized that expression of transcription factors that are required for induction and maintenance of pluripotency (Oct-4, Sox-2 and Nanog) would be a possible alternative to cell surface phenotype for identification of CSC populations. Therefore we evaluated Nanog expression in 4T1.2 murine breast carcinoma cells, as a biomarker for CSC sub-populations. Introduction of a Nanog promoter reporter that drives destabilized GFP into 4T1.2 cells resulted in a stable expression of GFP in about 1% of the cells. These GFP+ cell could be easily isolated by FACS cell sorting. On subsequent in vitro cell culture of GFP+ populations, up to 80% of the cells became GFP- over a period of 4 weeks. This would be consistent with a subsequent differentiation of these cells. By contrast GFP- populations remained 100% GFP-. In vitro CSC surrogate assays show that Nanog-GFP+ cells produced more spheroids in soft agar and under non-adherent growth conditions. More importantly, in vivo cell transfer studies demonstrated that Nanog-GFP+ cells were more efficient at generating experimental lung metastases when compared to Nanog-GFP-cells. Interestingly Nanog-GFP+ cells were more resistant to the chemotherapeutic drugs paclitaxel and bortezomib. However targeting the extrinsic apoptosis pathway with a combination of bortezomib and agonist antibodies to the TRAIL death receptor DR5 was equally effective against both Nanog-GFP+ and Nanog-GFP- cells. These findings suggest that Nanog promoter activity is a robust marker of highly metastatic 4T1.2 CSC subpopulations, and suggests that eradication of such cells may be required for improved anti-cancer therapies. Funded by NCI Contract HHSN261200800001E Citation Format: Alan D. Brooks, Rachel L. de Kluyver, Jimmy K. Stauffer, Marcella Kaddoura, Thomas J. Sayers. Using Nanog expression to identify breast cancer stem cell populations. [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 2656. doi:10.1158/1538-7445.AM2013-2656

New views and new tools for complex diseases

The non-transmittable chronic diseases such as cancer, cardiovascular, metabolic and neurodegeneration diseases already become the major threats to the mankind. All of these chronic diseases are recognized as complex diseases, which involve in multiple genetic factors, environment factors and the interaction of these two kinds of the factors, and the complicated molecular networks consisting of huge number of the genes and proteins. Therefore, it is clear that the conventional experimental biology that focuses on individual gene or protein is not enough to reveal the molecular mechanisms of complex diseases. The collection of six papers in this issue deliver new views to understand the molecular mechanisms of the initiation and progression of the complex diseases, and might provide new powerful tools to fight these diseases. Conventional way often labels particular genes as ‘oncogene’ or ‘tumor suppressor gene’ for their tumorigenic potential. Wu’s group revealed that a famous tumor suppressor gene p53 activated by 5-aza-2 ′ -deoxycytidine (ZdCyd) resulted in high protumorigenicity of mouse P19 embryonic carcinoma cells that are growing in suspension condition, whereas the ZdCyd-induced p53 activation resulted in suppression of tumorigenic potential of P19 EC cells growing on adherent condition. Based on their developed computational method, the authors revealed two different p53-dominant gene-regulatory networks of ZdCyd-treated P19 EC cells at adherent and non-adherent growth conditions, respectively. This study implies that changing cellular contexts of mammalian cells from adherent to non-adherent growth conditions rewires p53-regulating networks that could convert the tumor suppression to tumor-promoting activity. In addition, Auberger’s group reported that Imatinib, an inhibitor of a chimeric tyrosine kinase BCR-ABL, as the leading compound to treat chronic myelogenous leukemia (CML), presented various inhibitory effects on different CML cells. They also found that the variation in the drug effects was derived from different molecular contexts in the CML subpopulations. Regenerative medicine is an important way to treat the chronic diseases such as heart disease, diabetes and Alzheimer’s disease. Stem cells and the related molecular mechanisms of cell reprogramming have been recognized as potential tools for regenerative medicine. Asuelime and Shi reviewed the recent progress on cell-lineage reprogramming and its potential in regenerative medicine. Although the researchers have achieved significant progress, these authors conclude that more studies on this area need to be done before the technology based on lineage reprogramming is ready to the clinical practice. In addition, Kumar’s group in a letter reported that a transcription factor, zinc-finger transcription factor in cerebellum-3 (Zic3), combined with other three transcriptional factors, could induce the conversion of human fibroblasts to neuroprogenitorlike cells in a rapid way; and they suggest that this new approach to reprogramming cells could be a potential tool for screening chemical compounds against neural diseases. Diabetes is a typical age-dependent chronic disease. In this issue, Lammert’s group reported that the up-regulation of an antioxidant protein, DJ-1, in mouse and human islets during aging with non-diabetic conditions, whereas the expression of DJ-1 could be reduced in pancreatic islets of patients with type 2 diabetes mellitus (T2DM). Their further experimental data suggest that DJ-1 might serve as a novel drug target for T2DM. In another paper in this issue, Jia’s group showed that ficolin-3 as an innate immune protein presented a low serum level in the population with insulin-resistance based on a human cross-sectional study. The authors further proposed that serum ficolin-3 could be used as a biomarker for predicting the development of T2DM and even as a potential drugtarget for the treatment of T2DM. We hope that these papers will provide different angles from conventional views to understand the molecular mechanism of complex diseases and new potential tools against these diseases.

Abstract 4261: Epigenetic reprogramming affects malignant properties of human liver cancer cells

Abstract Background: A plastic phenotype is a significant hallmark of aggressive tumors and embryonic stem cells (ESC). Cancer plasticity is dynamically regulated both by cell microenvironment and epigenetic machinery therefore playing important roles in transcriptional reprogramming and cell fate stable changes. Objective: We have explored the relationship between epigenetic alterations and local microenvironment in regulation of cancer stem cells (CSCs) using Zebularine (Zeb), a DNA-methyltransferase1 inhibitor, in combination with the modulation of cellular density in culture. Methods: Seven human hepatoma cell lines, including two primary HCC cell lines, were plated at high (HD) and low density (LD) and grown in the presence or absence of Zeb for 3 days. After, cells were propagated in serum-free condition for evaluation of sphere-forming capacity. Primary tumor-spheres were passaged every 10 days to produce generation 1 (G1) trough G5. Spheres at different generations were examined for differences in gene expression and tumorigenic capacity in vivo. The sphere-derived mRNA and microRNA gene expression signatures generated for different passages were integrated with human HCC database and tested for their prognostic value by Kaplan-Meier. Results: Cells grown under LD failed to form spheres when placed in non-adherent growth conditions. Cells cultured at HD displayed a sphere forming potential at G1 but were unable to maintain extended sphere forming capacity regardless of Zeb pre-treatment. In contrast, Zeb-treated LD (LDZ) cells showed unique features in terms of self renewal potential both in vitro and vivo. LDZ-derived spheres were more proliferative as indicated by Ki67 staining whereas HDZ-spheres showed activation of apoptotic pathway and dowregulation of NF-kB protein levels. LDZ also maintained a higher expression of CSC related markers (CD133, CD44, EpCAM) as well as key factors involved in ESC self-renewal and pluripotency (Bmi1 and αSuz12, subunit of distinct polycomb complexes, PRC1 and PRC2). Also, LDZ-derived spheres consistently expressed higher levels of DNMT1 and DNMT3a. In addition, LDZ spheres exhibited epithelial-mesenchymal transition attributes as judged by the upregulation of β-catenin, ZEB1, VIM and SNAL1 markers as compared to HD spheres. Finally, only G1 LDZ-spheres derived cells showed higher in vivo tumorigenicity and could give rise to brain metastasis. Consistently, global transcriptome analysis confirmed that LDZ sphere signature was enriched in oncogenic molecules associated with metastasis, invasion and resistance to apoptosis, including SMAD2, CD133, CD44 and NF-kB and was able to predict the clinical outcome of liver cancer patients. Conclusions: Epigenetic reprogramming of liver cancer cell lines induced by a combined modulation of DNA methylation and cellular microenviroment generates long lasting memory effects changing the CSC malignant capacity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4261. doi:1538-7445.AM2012-4261

Characterisation of FAP-1 expression and CD95 mediated apoptosis in the A818-6 pancreatic adenocarcinoma differentiation system

The present study investigated the expression and localisation of FAP-1 (Fas associated phosphatase-1) and CD95 in a 3D differentiation model in comparison to 2D monolayers of the pancreatic adenocarcinoma cell line A818-6. Under non-adherent growth conditions, A818-6 cells differentiate into 3D highly organised polarised epithelial hollow spheres, resembling duct-like structures. A818-6 cells showed a differentiation–dependent FAP-1 localisation. Cells grown as 2D monolayers revealed FAP-1 staining in a juxtanuclear cisternal position, as well as localisation in the nucleus. After differentiation into hollow spheres, FAP-1 was relocated towards the actin cytoskeleton beneath the outer plasma membrane of polarised cells and no further nuclear localisation was observed. CD95 surface staining was found only in a subset of A818-6 monolayer cells, while differentiated hollow spheres appeared to express CD95 in all cells of a given sphere. We rarely observed co-localisation of CD95 and FAP-1 in A818-6 monolayer cells, but strong co-localisation beneath the outer plasma membrane in polarised cells. Analysis of surface expression by flow cytometry revealed that only a subset (36%) of monolayer cells showed CD95 surface expression, and after induction of hollow spheres, CD95 presentation at the outer plasma membrane was reduced to 13% of hollow spheres. Induction of apoptosis by stimulation with agonistic anti-CD95 antibodies, resulted in increased caspase activity in both, monolayer cells and hollow spheres. Knock down of FAP-1 mRNA in A818-6 monolayer cells did not alter resposiveness to CD95 agonistic antibodies. These data suggested that CD95 signal transduction was not affected by FAP-1 expression in A818-6 monolayer cells. In differentiated 3D hollow spheres, we found a polarisation-induced co-localisation of CD95 and FAP-1. A tight control of receptor surface representation and signalling induced apoptosis ensures controlled removal of individual cells instead of a “snowball effect” of apoptotic events.

Targeted inhibition of telomerase activity combined with chemotherapy demonstrates synergy in eliminating ovarian cancer spheroid-forming cells

ObjectiveTelomerase activity (TA) is often used as a molecular marker for cancer aggressiveness. Our objectives were to determine the TA in ovarian cancer cell lines and the effectiveness of targeting telomerase for cancer therapy. MethodsOvarian cancer cell lines of various histologic subtypes were chosen to correspond to decreasing levels of clinical aggressiveness. Cells were grown in non-adherent growth conditions to form spheroid-forming cells (SFC). Telomerase activity was quantified using the TRAPeze RT Telomerase Detection Kit and confirmed with luciferase reporter plasmid containing promoter of human telomerase reverse transcriptase (hTERT). Cell proliferation survival assays were performed after treatment with a small molecule telomerase inhibitor BIBR1532 both with and without multiple chemotherapeutic agents. ResultsCompared to monolayer, TA from SFC correlated to the innate clinical aggressiveness of ovarian cancer cell lines ES2, SKOV3, and TOV112D. Treatment with BIBR1532 resulted in up to a 12-fold decrease in TA compared to controls. SFCs were significantly more resistant to BIBR1532 compared to monolayer cell lines; however, it showed reasonable efficacy at 100uM. In combination assays, the addition of BIBR1532 to carboplatin yielded the most favorable results in regards to synergy in all three cell lines evaluated. ConclusionsTelomerase activity appears to correlate to the clinical aggressiveness seen in histologic subtypes of ovarian cancer. BIBR1532 demonstrated significant inhibition of TA as well as reasonable efficacy as a single agent. Inhibition of telomerase with BIBR1532 in combination with carboplatin demonstrated a more than additive effect in-vitro and could represent a novel targeted therapy for ovarian cancer.

Hedgehog signaling pathway regulates the growth of ovarian cancer spheroid forming cells

The hedgehog (Hh) pathway has been shown to be activated in numerous malignancies as well as in cancer stem cells. We sought to determine the importance of the Hh pathway in regulating growth and development of ovarian cancer spheroid-forming cells (SFCs). Ovarian cancer cell lines (ES2, TOV112D, OV90, and SKOV3) as well as a normal ovarian epithelial cell line (IOSE80) were grown in non-adherent growth conditions to form SFCs. Western blot analysis was used to determine the expression of Hh pathway proteins SMOH, PTCH, GLI1. SFCs were treated with Hh agonists (SHH and IHH) as well as an Hh inhibitor (cyclopamine) to determine changes in spheroid growth and survival. All ovarian cancer cell lines readily formed spheroids in non-adherent growth conditions while IOSE80 failed to form SFCs. Compared to IOSE80, ovarian cancer cell lines demonstrated significant activation of the Hh pathway as defined by increased expression of intranuclear GLI1. Both Hh agonists demonstrated significant increases in spheroid volume of at least 42-fold for SHH-treated cells and 46-fold for IHH-treated cells. With regard to survival, SFCs were 30-50% more resistant to cyclopamine than their corresponding monolayer cells. Despite this resistance, inhibition of the Hh pathway with cyclopamine prevented further growth of SFCs with a 10-, 5-, and 4-fold restriction in growth for ES2, SKOV3, and TOV112D, respectively. The hedgehog pathway appears to be important in regulating growth of ovarian cancer spheroid-forming cells. The activation and inhibition of this pathway demonstrates significant correlation to enhanced growth and growth restriction, respectively.

Abstract 2460: Tumorigenic potential is independent of sphere phenotype in liver cancer

Abstract Background: A phenotypic plasticity, a hallmark of embryonic development and cancer progression/metastasis, is dynamically regulated by extrinsic signals produced by niche and intrinsic cell autonomous mechanisms. Consequently, both cell microenvironment and genetic/epigenetic machinery play important roles in cell fate reprogramming. Objective: In this study we aimed to evaluate the effects of environmental and/or epigenetic modulation on cancer stem cell (CSC) properties using a panel of human HCC cell lines. Methods: Seven human hepatoma cell lines, including one primary HCC cell line, were plated at high (HD) and low density (LD) and grown in the presence or absence of DNA-methyltransferase inhibitor zebularine (Zeb) for 3 days. Thereafter cells were propagated in serum-free condition for evaluation of sphere-forming ability. Primary tumor-spheres were passaged every 10 days to generate passage 1 (P1) trough P7 spheres. Gene expression was examined by qRT-PCR and microarray analysis. Sphere tumorigenic capacity was evaluated in vivo after subcutaneous (s.c.) transplantation into NOD/SCID mice. Results: Cells grown under LD failed to form spheres when placed in non-adherent growth conditions. In contrast, Zeb-treated LD (LDZ) cells were capable of forming clonal-expanding spherical colonies. Cells cultured at HD possessed a comparable sphere forming ability regardless of Zeb pre-treatment. However, despite the similar phenotype and size, spheres formed by LDZ and HD/HDZ cells possessed different self-renewal and tumorigenic potential in vitro and in vivo. Thus, LDZ spheres exhibited a considerably stronger induction of the stem cell-related genes (NANOG, OCT4, and BMI1) as well as epithelial-mesenchymal transition markers (β-catenin, ZEB1, VIM and SNA1) as compared to HD spheres. Furthermore, in serial sphere formation assay, only LDZ-spheres could retain a long term self-renewal while the sphere-forming ability of both HD- and HDZ-spheres was exhausted within the first three passages in vitro. In addition, cells dissociated from LDZ-spheres at P1 possessed higher tumorigenicity upon s.c. transplantation into NOD/SCID mice as compared to P1 HD-spheres, and only LDZ-spheres could propagate the tumor growth in vivo at later passages. Conclusion: We describe functional tumor-sphere approach which offers an efficient tool to enrich for cells with CSC properties. By modulating either cell density and/or epigenome in hepatoma cell lines we selected for sphere-forming phenotype possessing a high tumor-initiating potential including a long-term ability for self-renew during serial sphere passaging and propagation of tumor growth in vivo. The global gene expression analysis is underway to characterize the molecular mechanisms responsible for maintaining CSC compartment in sphere-derived tumor clones. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2460. doi:10.1158/1538-7445.AM2011-2460

Abstract 3349: Nanog-expressing breast cancer tumor initiating cells (TICs) are highly metastatic and are resistant to the effects of proteasome inhibition

Abstract Functional heterogeneity of transformed cells exists within a single tumor mass and between distinct tumor nodules. Many tumors have also been demonstrated to contain a subpopulation of tumor initiating cells (TICs) whose epigenetic modifications convey resistance to conventional therapies. It has been hypothesized that TICs represent the terminal product of epithelial-mesenchymal transition which is linked to metastatic potential. Therefore we examined the metastatic potential of TICs in the 4T1 murine breast carcinoma model, and evaluated Nanog expression as a biomarker for highly metastatic 4T1 sub-populations. Traditionally TICs have been identified and isolated through expression of selected cell surface markers and functional assays, however cell surface phenotype alone fails to identify CSC in all tumors from the same organ or in all patients. We hypothesized that expression of transcription factors that are required for induction and maintenance of pluripotency (Oct-4, Sox-2 and Nanog) could be superior to cell surface phenotype for identification of TICs. Thus we adapted a commercial promoter reporter system where the Nanog promoter drives copepod GFP which has been destabilized to better reflect rapid changes in transcriptional activity. I ntroduction of a Nanog promoter reporter construct into 4T1.2 cells resulted in stable expression of GFP in about 0.5% of the cells. In vivo tumor transfer studies confirmed that Nanog promoter activity (4T1.2-GFPpos cells) was associated with a significantly enhanced ability to form experimental lung metastases. In addition, in vitro TIC surrogate assays showed that 4T1.2 GFPpos cells produced increased numbers of spheroids in both soft agar and under non-adherent growth conditions. Finally Nanog-GFPpos cells were more resistant to killing by Paclitaxel than Nanog-GFPneg cells, but were more sensitive to Salinomycin. Together these findings suggest that Nanog promoter activity is a robust marker of metastatic 4T1.2 TICs. We have previously observed that administration of the proteasome inhibitor Bortezomib (Bzb) has moderate anti-4T1.2 tumor activity in vivo therefore we examined the sensitivity of the various 4T1.2 populations to Bzb treatment in vitro. 4T1.2 Nanog-GFPpos cells were resistant to the cytostatic effects of Bzb while 4T1.2 Nanog-GFPneg cells were sensitive. The molecular basis of this difference is under investigation, but is not due to differences in accumulation of ubiquitinated proteins. Taken together, these findings show that Nanog promoter activity is a marker of highly metastatic TICs, and suggest that eradication of TICs may be required for curative anti-cancer therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3349. doi:10.1158/1538-7445.AM2011-3349

Abstract 1450: Loss of the UPR member CREB3L1 is vital for the survival of breast cancer cells in the tumor microenvironment and development of the metastatic phenotype

Abstract The unfolded protein response (UPR) is required for proper protein folding in the endoplasmic reticulum under conditions of stress such as those encountered in the tumor microenvironment. Recently, a number of studies have suggested a role for the unfolded protein response in the development of cancer, more specifically in regulating the balance between cell death, dormancy and the growth of cancer cells in the tumor microenvironment. This study examined the role of the transcription factor CREB3L1, a member of the UPR machinery, in breast cancer development and metastasis. Northern blotting analysis identified a loss of CREB3L1 expression in metastatic breast cancer cell lines compared to low- or non-metastatic cell lines, which was subsequently shown, through bisulphate sequencing, to be the result of enhanced promoter methylation. When metastatic cells were transfected with CREB3L1 they demonstrated a significant decrease in survival in hypoxic and non-adherent growth conditions, and reduced in vitro invasion and migration in chemotaxis assays. Interestingly, in an in vivo rat mammary tumor model where rats were injected with cells in the hind footpad, the CREB3L1 expressing rat cells not only failed to form metastases but tumor regression was seen in 70% of the animals. In contrast, metastases were seen in the popliteal lymph node of 90% of rats injected with wild-type and empty vector-transfected cells. Microarray and ChIP on Chip analyses identified changes in the expression of many genes involved in cancer development and metastasis, including a decrease in those involved in angiogenesis when CREB3L1 was expressed. We also observed a significant decrease in the number of migrating endothelial cells in scratch and chemotaxis assays in media conditioned by CREB3L1 expressing cells, compared to conditioned media from untransfected cells. To determine if these findings translated to human breast cancer, real-time PCR analysis of CREB3L1 expression in human breast cancer tissues of differing grade (n=30) was performed. Expression of CREB3L1 was elevated in low and medium grade tissue but substantially reduced in high grade tissue, compared to normal. The high grade breast cancer tissues demonstrated a 6.6 fold decrease in the expression of CREB3L1 compared to the low grade tissues (p<0.001), with about a 2 fold decrease compared to normal tissue. In conclusion, CREB3L1 expression is elevated in low and medium grade breast cancer tissue, perhaps as the cancer cells respond to various stresses. However, loss of CREB3L1 expression is required for the long-term survival of breast cancer cells in the tumor microenvironment and the progression to a highly metastatic phenotype. CREB3L1 expression may be a useful marker in predicting tumor grade and patient prognosis, in addition to providing a therapeutic target. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1450. doi:10.1158/1538-7445.AM2011-1450

Abstract 4297: Selective enrichment of liver cancer stem cell by DNA methyltransferase inhibition

Abstract Background: Evidence is growing that many solid cancers, including liver cancer, contain small subpopulations of cells with stem cell-like properties known as cancer stem cells (CSCs) that are responsible for maintaining and propagating a tumor. Major obstacles in the research on CSC biology are the paucity and lack of specific markers of CSC. We have recently shown that human liver CSC can be enriched by modulating the epigenome of liver cancer cell lines with zebularine (Zeb), a potent DNA methyltransferase inhibitor. Objective: In this study we exploited the impact of epigenetic modulation of cancer epigenome by Zeb in conjunction with the ability of stem/progenitor cells to grow as non-adherent spheres in serum-free media to provide further enrichment of the CSC fraction. Methods: Huh7 cells were plated in high (HD, 13×103cell/cm2) and low (LD, 2.7×103/cm2) density and treated with 100 µM Zeb for 3days. The sphere forming ability and serial sphere formation were investigated in Matrigel and suspension culture, respectively. CSC and pluripotency markers preferentially expressed in cells grown under sphere conditions were examined by qRT-PCR and surface markers by FACS. Results: Huh7 cells grown under LD failed to form tumorspheres when placed in Matrigel or non-adherent growth conditions. In striking contrast, Zeb-treated low density cells were capable of forming spherical clonal-expanding colonies which showed the stem cell-like properties such as strong induction of the stem cell-related genes NANOG and OCT4 and did not express EpCAM. Cells grown at high density possessed a similar sphere forming ability regardless of Zeb pretreatment. However, morphological characterization revealed a predominance of smaller sized spheres suggesting reduced proliferation potential and/or differences in cell type of origin, with smaller spheres originating from the progenitors, and larger spheres from the stem cells. Consistent with this, HD spheres showed a smaller induction of NANOG and OCT4, expressed high levels of EpCAM and exhibited reduced self-renewal in serial sphere formation assay. The sphere forming ability of LD and HD cells was associated with a presence of a subpopulation of CD133-/cKit+ cells. Conclusion: The described “double selective” approach offers an in vitro model to enrich for cells with cancer stem cell properties. Both cell density and epigenetic modulation were able to affect the sphere-forming potential of Huh7 cells. Further studies are needed to characterize this cell population in more detail, to clarify their tumorigenic potential and the capacity to propagate tumor growth in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4297.

Abstract 3334: Telomerase activity corresponds to the level of aggressiveness of ovarian cancer stem cells and are a potential site of targeted therapy

Abstract Objective: Cancer stem cells possess the important property of self-renewal. Telomerase activity (TA) is often used as a molecular marker for cancer aggressiveness and allows propagation of cancer cells by adding telomere repeats which can result in unlimited cell replication. As such, the potential of telomerase in maintaining self-renewal properties of cancer stem cells have been proposed. Our objectives were to determine the TA of ovarian cancer stem cells (OSC) and the effectiveness of targeting telomerase for cancer therapy. Methods Ovarian cancer cell lines (ES2, TOV112D, and SKOV3) were grown in non-adherent growth conditions to form spheroid forming cells for numerous successive spheroid generations. Flow cytometry determined the CD44+/CD24- phenotype consistent with cancer stem-cells. Histologic subtypes were chosen to correspond to decreasing levels of aggressiveness: ES2- clear cell; SKOV3- papillary serous; and TOV112D- endometrioid. TRAPEZE RT Telomerase Detection Kit utilized flourescently labeled primers to detect and quantify telomerase activity by RT-PCR. Results were confirmed with luciferase reporter plasmid containing promoter of human telomerase reverse transcriptase (hTERT). Each cancer cell line was treated with 10, 20, 50, and 100uM of a small molecule telomerase inhibitor BIBR1532 measured by cell proliferation survival assay for a total of 72 hours. Results All ovarian cancer cell lines readily formed spheroids in non-adherent growth conditions with flow cytometry demonstrating the enrichment of cancer stem cell phenotype: CD44+/CD24-. TA was significantly increased in spheroid forming cells enriched for OSC in both ES2 (5-fold increase) and SKOV3 (6-fold increase) (p < 0.001), however TOV112D showed a decrease in TA. Similarly, ES2 and SKOV3 showed significant TA by luciferase assay with 400% and 1,700%, respectively (p < 0.001), while TOV112D had a decrease in TA. In all cell lines studied, treatment with BIBR1532 demonstrated significant cell death in a dose-dependent fashion with a 5-fold cell kill and only 20% of cancer cell survival at 72 hours. Conclusions Telomerase activity appears to increase with ovarian cancer stem cells and supports the hypothesis that it could be important in the self-renewal properties of cancer stem cells. Inhibition of telomerase with a competitive small molecule demonstrated significant response in-vitro and could represent novel targeted therapy for ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3334.

Human CD34+/CD90+ ASCs Are Capable of Growing as Sphere Clusters, Producing High Levels of VEGF and Forming Capillaries

BackgroundHuman adult adipose tissue is an abundant source of mesenchymal stem cells (MSCs). Moreover, it is an easily accessible site producing a considerable amount of stem cells.Methodology/Principal FindingsIn this study, we have selected and characterized stem cells within the stromal vascular fraction (SVF) of human adult adipose tissue with the aim of understanding their differentiation capabilities and performance. We have found, within the SVF, different cell populations expressing MSC markers – including CD34, CD90, CD29, CD44, CD105, and CD117 – and endothelial-progenitor-cell markers – including CD34, CD90, CD44, and CD54. Interestingly, CD34+/CD90+ cells formed sphere clusters, when placed in non-adherent growth conditions. Moreover, they showed a high proliferative capability, a telomerase activity that was significantly higher than that found in differentiated cells, and contained a fraction of cells displaying the phenotype of a side population. When cultured in adipogenic medium, CD34+/CD90+ quickly differentiated into adipocytes. In addition, they differentiated into endothelial cells (CD31+/VEGF+/Flk-1+) and, when placed in methylcellulose, were capable of forming capillary-like structures producing a high level of VEGF, as substantiated with ELISA tests.Conclusions/SignificanceOur results demonstrate, for the first time, that CD34+/CD90+ cells of human adipose tissue are capable of forming sphere clusters, when grown in free-floating conditions, and differentiate in endothelial cells that form capillary-like structures in methylcellulose. These cells might be suitable for tissue reconstruction in regenerative medicine, especially when patients need treatments for vascular disease.

Cyclin A Is a c-Jun Target Gene and Is Necessary for c-Jun-induced Anchorage-independent Growth in RAT1a Cells

Overexpression of c-Jun enables Rat1a cells to grow in an anchorage-independent manner. We used an inducible c-Jun system under the regulation of doxycycline in Rat1a cells to identify potential c-Jun target genes necessary for c-Jun-induced anchorage-independent growth. Induction of c-Jun results in sustained expression of cyclin A in the nonadherent state with only minimal expression in the absence of c-Jun. The promoter activity of cyclin A2 was 4-fold higher in Rat1a cells in which c-Jun expression was induced compared with the control cells. Chromatin immunoprecipitation demonstrated that c-Jun bound directly to the cyclin A2 promoter. Mutation analysis of the cyclin A2 promoter mapped the c-Jun regulatory site to an ATF site at position -80. c-Jun was able to bind to this site both in vitro and in vivo, and mutation of this site completely abolished promoter activity. Cyclin A1 was also elevated in c-Jun-overexpressing Rat1a cells; however, c-Jun did not regulate this gene directly, since it did not bind directly to the cyclin A1 promoter. Suppression of cyclin A expression via the introduction of a cyclin A antisense sequences significantly reduced the ability of c-Jun-overexpressing Rat1a cells to grow in an anchorage-independent fashion. Taken together, these results suggest that cyclin A is a target of c-Jun and is necessary but not sufficient for c-Jun-induced anchorage-independent growth. In addition, we demonstrated that the cytoplasmic oncogenes Ras and Src transcriptionally activated the cyclin A2 promoter via the ATF site at position -80. Using a dominant negative c-Jun mutant, TAM67, we showed that this transcriptional activation of cyclin A2 requires c-Jun. Thus, our results suggest that c-Jun is a mediator of the aberrant cyclin A2 expression associated with Ras/Src-induced transformation.

HMG-I/Y Is a c-Jun/Activator Protein-1 Target Gene and Is Necessary for c-Jun–Induced Anchorage-Independent Growth in Rat1a Cells

Abstract The transcription complex activator protein-1 (AP-1) plays a role in a diverse number of cellular processes including proliferation, differentiation, and apoptosis. To identify AP-1–responsive target genes, we used a doxycycline-inducible c-Jun system in Rat1a cells. The HMG-I/Y chromatin binding protein was found to be up-regulated by c-Jun. Following induction of c-Jun expression, Rat1a cells under nonadherent growth conditions have sustained HMG-I/Y mRNA expression and 2-fold higher protein than uninduced cells. HMG-I/Y promoter reporter assays show that HMG-I/Y promoter activity increases in the presence of c-Jun expression, and gel mobility shift assays demonstrate that induced c-Jun binds to an AP-1 consensus site at position −1,091 in the HMG-I/Y promoter. Suppression of HMG-I/Y expression by its antisense sequence significantly reduces the ability of c-Jun–overexpressing Rat1a cells to grow in an anchorage-independent fashion. HMG-I/Y transforms Rat1a cells (although the colonies are smaller than that observed for the cells overexpressing c-Jun). Taken together, these results suggest that HMG-I/Y is a direct transcriptional target of c-Jun necessary for c-Jun–induced anchorage-independent growth in Rat1a cells.