Termed Cancer Stem Cells Research Articles

Wnt Antagonist, Secreted Frizzled-Related Protein 4 (sFRP4), Increases Chemotherapeutic Response of Glioma Stem-Like Cells

Malignant gliomas have a highly tumorigenic subpopulation, termed cancer stem cells (CSCs), that drives tumor formation and proliferation. CSCs possess inherent resistance mechanisms against radiation- and chemotherapy-induced cancer cell death, enabling them to survive and initiate tumor recurrence. We examined the effect of secreted frizzled-related protein 4 (sFRP4), a Wnt signaling antagonist, in chemosensitizing the glioma cell line U138MG and glioma stem cells (GSCs) enriched from U138MG to chemotherapeutics. We found that sFRP4 alone and in combination with either doxorubicin or cisplatin induced apoptosis. Proliferation decreased substantially in GSC-enriched population as measured by MTT and BrdU assays. JC-1 and caspase-3 assays demonstrated that cell death was through the apoptotic pathway. sFRP4 treatment also decreased neurosphere formation and induced neuronal differentiation. Inhibition by sFRP4 was abolished by Wnt3a, indicating that sFRP4 acts through the frizzled receptor. Further indication that sFRP4 acts through the Wnt β-catenin pathway was provided by decrease in the β-catenin protein and decrease in the β-catenin-stimulated gene cyclin D1 upon sFRP4 induction. By real-time PCR, an increase in apoptotic markers Bax and p21, a decrease in pro-proliferative marker CycD1, and a decrease in the GSC marker CD133 were observed. These observations indicate that sFRP4 is able to sensitize glioma cells and stem cells to chemotherapeutics. We thus identified for the first time that sFRP4 could help to destroy cancer stem cells of glioma cell line, which would lead to effective treatment regimen to combat brain tumors.

Micro RNA Regulation of Cancer Stem Cell Phenotypes

A small subpopulation of cancer cells, termed Cancer Stem Cells (CSCs), are primarily responsible for initiating metastasis, resistance to therapy and ultimately patient relapse. If any improvements in long- term patient survival are to be achieved, it is vital to target this highly tumorigenic population of cells. The cancer stem-like phenotype is defined by the capability to self-renew, differentiate and proliferate. A number of signaling pathways and stem cell markers have been demonstrated to regulate these vital activities, including Wnt/β- catenin, Hedgehog, Notch, BMI-1 and HMGA2. Intense interest is now focused on developing strategies to eliminate the CSC population. In this regard, several microRNAs (miRNAs), including miR-451, miR- 34a, miR-17-92 and miR-200, have been demonstrated to regulate these fundamental CSC signaling pathways and consequently modulate the CSC phenotype. Thus, modulation of CSC-related miRNAs may represent a plausible mechanism for targeting the CSC population. The pleiotropic nature of miRNA activity can be exploited in a therapeutic manner to simultaneously inhibit multiple key regulators of the CSC phenotype and potentially achieve more efficacious targeting. This article will review our current understanding of the role of miRNAs in regulating CSC signaling pathways. In particular, this review will provide a better understanding of the role of specific miRNAs in regulating the key CSC signaling pathways and highlight several miRNAs that could be therapeutically applicable to eliminate the CSC population.

The STAT3-Ser/Hes3 signaling axis in cancer

Disrupting the regenerative capacity of tumorigenic cells is a major focus in medicine. These regenerative properties are carried by a subpopulation of cells within the tumor, termed cancer stem cells. Current therapies don't effectively tackle the disease suggesting these cells employ yet unidentified molecular mechanisms allowing them to evade targeting. Recent observations in neural stem cells reveal an extraordinary plasticity in the signaling pathways they utilize to grow. These findings are being extended to the cancer stem cell field, illuminating conceptually novel treatment strategies. Tumorigenic cells can make use of distinct, even opposing pathways, including JAK/STAT and the non-canonical STAT3-Ser/Hes3 signaling axis. This plasticity may not be confined to the cancer stem cell population, but may be shared by various cell types within the tumor, blurring the line distinguishing cancer stem cells from other tumor cell types. The implications to anti-cancer medicine are highly significant, since these findings demonstrate that inhibiting one cell growth pathway may actually enhance the activity of alternative ones. Drug discovery programs will also benefit from these concepts.

Evaluation of multiple stemness markers in normal thyroid and in thyroid carcinoma (TC) specimens.

The tumorigenic capacity in cancer, including TC, is confined in a small subpopulation of stem-like cells termed cancer stem cells (CSCs). To date, markers for thyroid CSCs remain to be confirmed. Thus, the identification of a specific antigen to distinguish thyroid CSCs from normal SCs represents a crucial step to develop targeted therapies. The aim of this study was to investigate the expression of stemness markers and to isolate putative SCs from both normal thyroid and TC. Normal and neoplastic tissue samples were harvested from a cohort of 19 patients affected by TC, who underwent thyroidectomy. Fresh specimens were enzymatically digested. Cell suspensions were characterized by flow cytometry using anti-CD133, -CD45, -CD31, -CD44 and -CD105 antibodies. A portion of cells was suspended in SC medium (SCM) in order to obtain thyroid spheres. CD133+ and CD105+ cell rates were significantly higher in TC than in normal thyroid tissue (2.5% vs. 0.1% and 3.9% vs. 0.5% respectively, p<0.02). No difference was found regarding CD44+. In TC both CD45+ and CD31+ cell rates were 1.3%, but 1.45% of CD133+ cells were also positive for CD45 and 0.8% for CD31. In normal tissues CD45+ and CD31+ cell rates were 0.5% and 0.03%, respectively, but none of CD133+ cells were positive for CD45 and CD31. Putative SCs were isolated particularly from TC samples; they grew as both freefloating spheres and adherent cells. The re-evaluation of markers in putative SC isolated from 2 cases of normal tissue showed that cells expressed higher levels of CD133, CD105 and CD44 with respect to those obtained after tissue digestion. These preliminary findings suggest the presence of a higher fraction of CD133+ and CD105+ cells in TC samples compared to normal tissues. The higher expression of the stemness markers in TC may be related to the better survival of cells obtained from the TC samples in the SCM. Further investigation is required to draw any conclusion, but our approach seems to be promising.

Prolyl‐isomerase Pin1 controls normal and cancer stem cells of the breast

Mammary epithelial stem cells are fundamental to maintain tissue integrity. Cancer stem cells (CSCs) are implicated in both treatment resistance and disease relapse, and the molecular bases of their malignant properties are still poorly understood. Here we show that both normal stem cells and CSCs of the breast are controlled by the prolyl-isomerase Pin1. Mechanistically, following interaction with Pin1, Notch1 and Notch4, key regulators of cell fate, escape from proteasomal degradation by their major ubiquitin-ligase Fbxw7α. Functionally, we show that Fbxw7α acts as an essential negative regulator of breast CSCs' expansion by restraining Notch activity, but the establishment of a Notch/Pin1 active circuitry opposes this effect, thus promoting breast CSCs self-renewal, tumor growth and metastasis in vivo. In human breast cancers, despite Fbxw7α expression, high levels of Pin1 sustain Notch signaling, which correlates with poor prognosis. Suppression of Pin1 holds promise in reverting aggressive phenotypes, through CSC exhaustion as well as recovered drug sensitivity carrying relevant implications for therapy of breast cancers.

Abstract A54: Isolation and characterization of cancer stem cells with peptides.

Abstract Background: Cancer as the most malignant disease still demands the more effective therapeutics methods although continuous efforts have been input to cancer therapies. Recent studies indicate that only a small subset of cancer cells termed cancer stem cells (CSCs) contributes to tumor progression, metastasis and drug-resistance. Drugs targeting to CSCs might become more effective approaches to cancer therapy. To date, isolation and characterization of CSCs have largely depended on surface markers which are not specific or well-recognized for individual tumor types. Our goal is to develop methods for isolating and characterizing a set of lung CSCs based on the specific binding peptides for CSCs without any recognized definitive specific surface markers. Materials and Methods: Stem-like cells were enriched from non-small cell lung cancer (NSCLC) cell line H460 by sphere-forming assay. Characterized these stem-like cells by colony-forming assay, drug-resistance assay and soft agar assay. Screening and identification of specific binding peptides for CSCs were performed with bacteria surface display methods. The affinity between the specific binding peptides and CSCs was determined with fluorescence microscope and flow cytometric analysis. The specificity of peptides binding with CSCs was examined through comparing the binding capacity of peptides with either H460 cells or other cell lines including stem cells. The isolation of CSCs was performed using specific fluorescent peptides with FACS machine. The further characterization of selected CSCs including exploring the source and tumorigenesis mechanism of CSCs was performed through miRNA analysis. Results: Stem-like cells divided and formed tumor spheres in a couple of weeks, while most of cancer cells died in the serum-free medium. These stem-like cells were able to grow indefinitely as tumor spheres in long-term culture (more than 20 passages). Meanwhile, sphere cells could form colonies in the soft agar plates and exhibit the drug resistance capacity, which indicated that these sphere cells behaved as CSCs. Three peptides binding specifically to those CSCs were identified with bacteria surface display method. They could seldom bind with other cell types including H460 cells, H1299 sphere cells, HLF cells, MSCs and ESCs. One selected peptides named HCBP-1 exhibited highly specific binding capacity to H460 CSCs. The apparent dissociation constant (KD) was 6.4 × 10-7 M with a flow cytometry-based assay. In the peptides binding subgroup of H460 cells, at least 10 miRNAs presented the different expression rate from the H460 cells. Conclusions: In our study, a panel of specific binding peptides was identified with bacteria surface display method which could be used for isolation and characterization of CSCs without definitive CSCs markers. Furthermore, this new method could be potentially expanded to characterize and isolate CSCs in any types of cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A54. Citation Format: Yimin Zhu, Anxin Wang. Isolation and characterization of cancer stem cells with peptides. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A54.

Pien Tze Huang suppresses the stem-like side population in colorectal cancer cells

Accumulating evidence suggests that a small population of cells termed cancer stem cells (CSCs) are crucial in tumor development and drug resistance, leading to cancer relapse and metastasis and eventually the failure of clinical cancer treatment. Therefore, targeting CSCs is a promising approach for anticancer therapies. Due to the drug resistance and adverse effects of currently used chemotherapies, traditional Chinese medicines (TCM) have recently received attention due to the relatively few side-effects. Thus, they have been used as important alternative remedies for various diseases, including cancer. Pien Tze Huang (PZH), a well-known TCM formula that was first prescribed more than 450years ago in the Ming Dynasty, has been used in China and Southeast Asia for centuries as a folk remedy for various types of cancer. Previously, it was reported that PZH inhibits colon cancer growth via the promotion of cancer cell apoptosis and inhibition of cell proliferation and tumor angiogenesis, which is probably mediated by its regulatory effect on multiple intracellular signaling pathways. To elucidate the mechanism of the tumoricidal activity of PZH, the aim of the present study was to investigate the effect of PZH on CSCs that were isolated as the side population (SP) from the HT-29 colorectal cancer cell line. The results demonstrated that PZH significantly and dose-dependently reduced the percentage of the colorectal cancer stem-like SP cells, decreased the viability and sphere-forming capacity of HT-29 SP cells, indicating that PZH is potent in suppressing the growth of colorectal cancer stem cells. Moreover, PZH treatment in HT-29 SP cells markedly inhibited the mRNA levels of ABCB1 and ABCG2, which are members of the ABC transporter superfamily, thereby contributing to the SP phenotype and multi-drug resistance. Findings of the present study suggest that inhibiting the growth of CSCs is a potential mechanism by which PZH can be used in cancer treatment.

Deciphering the role of stroma in pancreatic cancer.

This review intends to describe recent studies on pancreatic tumor-associated stroma and potential opportunities and limitations to its targeting. One of the defining features of pancreatic cancer is extensive desmoplasia, or an inflammatory, fibrotic reaction. Carcinoma cells live in this complex microenvironment which is comprised of extracellular matrix (ECM), diffusible growth factors, cytokines and a variety of nonepithelial cell types including endothelial cells, immune cells, fibroblasts, myofibroblasts and stellate cells. In addition to the heterogeneity noted in the nonneoplastic cells within the tumor microenvironment, it has also been recognized that neoplastic cancer cells themselves are heterogeneous, and include a subpopulation of stem-cell like cells within tumors termed cancer stem cells. Due to the failure of current therapeutics to improve outcomes in patients with pancreatic cancer, new therapeutic avenues targeting different components of the tumor microenvironment are being investigated. In this review article, we will focus on recent studies regarding the function of the tumor stroma in pancreatic cancer and therapeutic treatments that are being advanced to target the stroma as a critical part of tumor management. Recent studies have shed new light on the contribution of the pancreatic cancer fibroinflammatory stroma to pancreatic cancer biology. Additional studies are needed to better define its full contribution to tumor behavior and how to best understand the optimal ways to develop therapies that counteract its pro-neoplastic properties.

Cancer cells acquire a drug resistant, highly tumorigenic, cancer stem‐like phenotype through modulation of the PI3K/Akt/β‐catenin/CBP pathway

Cancer initiation and progression have been attributed to newly discovered subpopulations of self-renewing, highly tumorigenic, drug-resistant tumor cells termed cancer stem cells. Recently, we and others reported a new phenotypic plasticity wherein highly tumorigenic, drug-resistant cell populations could arise not only from pre-existing cancer stem-like populations but also from cancer cells lacking these properties. In the current study, we hypothesized that this newfound phenotypic plasticity may be mediated by PI3K/Akt and Wnt/β-catenin signaling, pathways previously implicated in carcinogenesis, pluripotency and drug resistance. Using GFP expression, Hoechst dye exclusion and fluorescence activated cell sorting (FACS) of cancer cell lines, we identified and tracked cancer stem-like side populations (SP) of cancer cells characterized by high tumorigenicity and drug resistance. We found that pharmacological inhibition or genetic depletion of PI3K and AKT markedly reduced the spontaneous conversion of nonside population (NSP) cells into cancer stem-like SP cells, whereas PI3K/Akt activation conversely enhanced NSP to SP conversion. PI3K/AKT signaling was mediated through downstream phosphorylation of GSK3β, which led to activation and accumulation of β-catenin. Accordingly, pharmacological or genetic perturbation of GSK3β or β-catenin dramatically impacted conversion of NSP to SP. Further downstream, β-catenin's effects on NSP-SP equilibrium were dependent upon its interaction with CBP, a KAT3 family coactivator. These studies provide a mechanistic model wherein PI3K/Akt/β-catenin/CBP signaling mediates phenotypic plasticity in and out of a drug-resistant, highly tumorigenic state. Therefore, targeting this pathway has unique potential for overcoming the therapy resistance and disease progression attributed to the cancer stem-like phenotype.

Epigenetic regulation of SOX9 by the NF-κB signaling pathway in pancreatic cancer stem cells.

Pancreatic cancer is the fourth leading cause of cancer-related mortality in the world. Pancreatic cancer can be localized, locally advanced, or metastatic. The median 1- and 5-year survival rates are 25% and 6%, respectively. Epigenetic modifications such as DNA methylation play a significant role during both normal human development and cancer progression. To investigate epigenetic regulation of genes in the tumor-initiating population of pancreatic cancer cells, which are also termed cancer stem cells (CSCs), we conducted epigenetic arrays in PANC1 and HPAC pancreatic cancer cell lines and compared the global DNA methylation status of CpG promoters in invasive cells, demonstrated to be CSCs, to their noninvasive counterparts, or non-CSCs. Our results suggested that the NF-κB pathway is one of the most activated pathways in pancreatic CSCs. In agreement with this, we determined that upon treatment with NF-κB pathway inhibitors, the stem cell-like properties of cells are significantly disrupted. Moreover, SOX9, demethylated in CSCs, is shown to play a crucial role in the invasion process. Additionally, we found a potential NF-κB binding site located in the SOX9 promoter and determined that the NF-κB subunit p65 positively regulates SOX9 expression by binding to its promoter directly. This interaction can be efficiently blocked by NF-κB inhibitors. Thus, our work establishes a link between the classic NF-κB signaling transduction pathway and the invasiveness of pancreatic CSCs, which may result in the identification of novel signals and molecules that function at an epigenetic level, and could potentially be targeted for pharmaceutical investigations and clinical trials.

Radiation resistance of cancer stem cells as an obstacle in cancer therapy

Sh Faisal 14.00 Radiation represents an important therapeutic intervention in many tumor types. Nevertheless, tumor recurrence continues to post major limitation to this type of therapy. Wealth of data suggests that there is a distinct population of cancer cells with exclusive ability to self-renew, termed cancer stem cells (CSCs). This subset of cancer cells has been shown to be resistant to conventional therapy, including chemotherapy and radiation. Failure to eradicate CSCs may result in tumor recurrence. This review article summarized the characteristic and identification of CSCs along with mechanisms of resistance in these cells including increased DNA repair. Understanding the mechanisms underlying radioresistance of CSCs will lead to better combination therapies to overcome resistance of these cells which will ultimately improve outcome for radiation treatment. Normal 0 false false false EN-US X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:Table Normal; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin:0in; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:Calibri,sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin;}

Expression of stem cell markers in pancreatic ductal adenocarcinoma and clinical relevance.

e15058 Background: Emerging evidence has suggested that malignant tumors are heterogeneous and that are composed of a small subset of distinct cancer cells (usually defined by cell surface marker expression) that are responsible for tumor initiation and propagation, termed cancer stem cells. These cancer stem cells (CSCs), or tumor-initiating cells (TICs), exhibit properties of normal stem cells and are associated with resistance to current therapies. It was found that human pancreatic cancer has shown a population of cancer stem cells that have aberrantly activated developmental signaling pathways, are resistant to standard chemotherapy and radiation, and have up-regulated signaling cascades that are integral for tumor metastasis. The aim of our study is to investigate the prognostic implication of cancer stem cell markers in pancreatic ductal adenocarcinoma. Methods: In 43 histological samples of pancreatic ductal adenocarcinoma were performed molecular biology assessment of CD 24, CD44, CD133, CD166, OCT3/4, LGR5. Results: At univariate analysis patients with an overexpression of CD44, CD133, OCT3/4 showed a worse prognosis in terms of overall survival (respectively: p=0.031; p=0.014; p=0.001). At multivariate analysis OCT3/4 resulted to be independent factor influencing outcome (HR=0.23). Patients with overexpression of all factors seem to have a worse OS compared to patients expressing only two, one or none (8.6 vs. 15.6 vs. 18.0 vs. 59.9 mts; p=0.006). Conclusions: Our data suggest that the presence of cancer stem cells could be linked with tumor aggressiveness and patients’ survival. This finding could drive therapeutic decision towards less or more intensive treatment.

Abstract 3743: Effect of folic acid supplementation on epigenetics and cancer stem cell populations in established colorectal cancer cell lines.

Abstract Mandated fortification of folic acid, a B vitamin involved in nucleotide synthesis and DNA CpG methylation, has led to a subsequent increase in blood folate concentration as well as a simultaneous increase in colorectal cancer incidence in North America. Recent findings suggest that a proportion of tumor cells, termed cancer stem cells (CSC), share properties with native stem cells and are vital in the development and perpetuation of tumor metastasis. Both epigenetic DNA modifications as well as the activation of established stem cell signaling pathways are responsible for the activation and maintenance of (CSC). CSC share properties with native stem cells and are vital in the development of primary lesions and the perpetuation of tumor metastasis. We hypothesize that folate, a CpG methylation altering agent, and extracellular microenvironment factors can influence the development and maintenance of CSCs. By isolating and characterizing the CSC fraction in established human colorectal cancer cell lines HCT116, Caco2, and SW480, LS174T and PC7, a subline of DLD-1, we have assessed the effect of folate on CSC signaling pathways and global DNA methylation. Cell lines were grown in suspension and supplemented with specialized stem cell media to promote the growth and expansion of the CSC fraction. Western blot analysis and immunofluorescence were used to characterize the protein expression and nuclear localization of proteins associated with established CSC maintenance pathways, respectively. Global DNA methylation status was assessed using an ELISA-based assay. HCT116 and SW480 show an increased ability to form colonospheres in culture compared to Caco2 and IEC18 (normal rat intestinal cell line). Cells grown in suspension show variable activation of stem cell renewal pathways compared to monolayer. Low folic acid levels led to reduced methylation status and colonosphere yield while high folic acid levels led to increased global methylation and an increased in colonosphere yield. Thus, altered folic acid exposure in vitro can influence the methylation status and CSC self-renewal ability of human colorectal cancer cells. The relationship between folic acid and cancer stem cell maintenance may be an important factor in the apparent duel modulatory effect of folic acid on colorectal cancer and warrants further investigation. Citation Format: Nathan Farias, Brenda Coomber. Effect of folic acid supplementation on epigenetics and cancer stem cell populations in established colorectal cancer cell lines. [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 3743. doi:10.1158/1538-7445.AM2013-3743

Abstract 3748: The Mre11/Rad50/Nbs1 (MRN) complex: A critical mediator of chemoresistance and cancer stem cell progression in head and neck squamous cell carcinoma.

Abstract While platinum-based agents like cisplatin (DDP) are the mainstay of chemotherapeutic options for several tumor types, including ovarian, lung, and head and neck squamous cell carcinoma, resistance to these agents can significantly contribute to treatment failure and increase the need for higher toxic doses. Tumors are heterogeneous in nature, with their growth sustained by a small subpopulation of highly malignant cells, termed cancer stem cells (CSCs). These cells comprise the top of the tumor cell hierarchy, are remarkably chemoresistant, and are associated with tumor recurrence. Enhanced repair of DNA double stand breaks (DSBs) may compensate for DDP induced damage and contribute to chemoresistance in tumor cells. The Mre11/Rad50/Nbs1 (MRN) complex is a key player in DNA DSB repair, telomere maintenance, and cell cycle checkpoint control, and has been associated with DDP resistance. The present study investigates the role of MRN in the development of a CSC-mediated chemoresistant phenotype and its associated molecular signature. Human head and neck squamous cell carcinoma (HNSCC) cell lines, a mouse model with human HNSCC, and tissue from HNSCC patients previously treated with DDP monotherapy were used in the study. In order to demonstrate the role of MRN on DDP resistance, two cell lines with significantly different sensitivity to DDP were chosen. In vitro and in vivo studies were performed to evaluate whether the chemoresistant cell line experienced increased CSC levels after DDP treatment, and whether enhanced MRN complex mediated DNA repair could explain this differential response. In addition, we assessed whether disrupting the MRN complex could lead to downregulation of these disparate effects on MRN and DNA repair, in both the CSCs and the rest of the tumor cells, conferring increased sensitivity to DDP. We evaluated both MRN expression and CSC levels, and how they correlated with apoptosis levels in patient tissue resected three days after a three day course of induction DDP monotherapy. Our study demonstrates that CSC levels and MRN expression strongly correlate with chemoresistance in vitro, in vivo, and in human. Importantly, disruption of MRN leads to increased sensitivity of HNSCC cells to DDP treatment. In conclusion, we propose to elucidate a novel mechanism responsible for CSC mediated chemoresistance, which relies on enhanced DNA DSB repair via upregulation of the MRN complex. Our findings emphasize that disrupting this complex leads to increased sensitivity of HNSCC cells to DDP, potentially reducing the need for high, toxic doses of DDP used clinically and improving treatment outcomes. Citation Format: Danish Nagda, Shayanne A. Lajud, Taku Yamashita, Sanjeev Kumar, Jun Zheng, Peter Qiao, Orysia Bezpalko, Bert W. O'Malley, Daqing Li. The Mre11/Rad50/Nbs1 (MRN) complex: A critical mediator of chemoresistance and cancer stem cell progression in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3748. doi:10.1158/1538-7445.AM2013-3748

Abstract 252: Molecular characterization of cancer stem cells isolated from primary colon cancer cells.

Abstract Colon cancer is the 3rd most common type of cancer and the 4th leading cause of cancer-related deaths worldwide. OncoTrack is an international consortium that has launched one of Europe's largest collaborative academic-industry research projects to develop and assess novel approaches for identification of new markers for colon cancer (1). To this end, colorectal tumors and metastases, as well as isolated circulating tumor cells and cancer stem cells are subjected to high-throughput sequencing of DNA and RNA. The laboratory data are combined with clinical data and used for in silico modeling of the patients' tumors and prediction of biomarkers as well as a tailored therapy. These predictions are validated using patient-specific in vitro (primary cells) and in vivo (xenografts) models. Recent data show that a small subpopulation of cells within different tumors, including colon cancer, is responsible for tumor growth and maintenance. Due to their functional similarities to adult tissue stem cells and their likely origin, these cells have been termed cancer stem cells. We aim at characterizing patient-derived primary cell cultures and cancer stem cells isolated from these. Primary tumors and metastases from colon cancer patients are cultivated in vitro using a matrigel-based, serum-free culture system. This system allows for long-term expansion of primary colon cancer cells in vitro (2). Using this methodology, we were able to establish over 35 novel, patient-specific colon cancer cell lines, both from primary tumors and metastases. Using previously established markers of colon cancer stem cells (CD44+, CD133+, CD166+, EpCAM-high, Wnt-high), cells are sorted by flow cytometry and subsequently subjected to genome-wide analyses using next-generation sequencing. These experiments allow for a detailed characterization of colon cancer stem cells at the level of transcriptome and methylome. It has been shown that tumors are heterogeneous with regard to gene mutations and expression of cancer stem cell markers (3). Using molecular tools such as padlock probes (4), we are analyzing the distribution of gene mutations identified by next-generation sequencing within primary cells as well as primary tumors. Moreover, we are addressing the expression pattern of cancer stem cell markers in these cell types. These data will help in a better understanding of individual tumors and assist in design of a personalized therapy. OncoTrack is a project funded by the Innovative Medicines Initiative Joint Undertaking (IMI JU). 1 Elsner M, Nature Biotechnology 29, 378 (2011); www.oncotrack.eu 2 Sato T el al., Gastroenterology. 2011 Nov;141(5):1762-72. 3 Gerlinger M et al., N Engl J Med. 2012 Mar 8;366(10):883-92. 4 Larsson C, et al., Nat Methods. 2010 May;7(5):395-7. Citation Format: Martin Lange, Dirk Schumacher, Thomas Hauling, Christian Regenbrecht, Oliver Politz, Mats Nilsson, Jens Hoffmann, Reinhold Schaefer, David Henderson. Molecular characterization of cancer stem cells isolated from primary colon cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 252. doi:10.1158/1538-7445.AM2013-252

Influence of the factors OCT3/4, SOX2, KLF4 or c-Myc on cell transformation promoted by the oncoprotein tax from HTLV-I.

Event Abstract Back to Event Influence of the factors OCT3/4, SOX2, KLF4 or c-Myc on cell transformation promoted by the oncoprotein tax from HTLV-I. Gustavo E. Brito-Melo1* 1 Federal University of the Jequitinhonha and Mucuri Valleys - UFVM, Pharmacy, Brazil Human T-cell leukemia virus type 1 (HTLV-I) infects approximately 15-20 milion people worldwide and is able to cause Adult T-cell Leukemia (ATL). The virus encodes the oncoprotein Tax wich can immortalize human T-lymphocytes and transform rodent cells in vitro. However the mechanism involved in cell tranformation by Tax are not fully understood. It has been shown that cancers are comprised of two broad categories of cells: those with high- and those with limited- proliferative potential. Cells in the former category are termed cancer stem cells. CSCs share several properties with adult stem cells, particularly the abilities to self-renew and differentiate into multiple cell types. Our new research proposal addresses a key question "Can the HTLV-1 Tax oncoprotein transform embryonic stem cells better than primary cells?” In the present study NIH3T3 cells were transfected with both Tax and individual pluripotence-associated factors (OCT3/4, SOX2, KLF4 and cMYC into CMV3.1 expression plasmid) communly used for cell reprograming. Hella cells were used as tranformed cell control. The cell growing of the transfected cells was kept on soft agar and the cell colony counting (UFC) was achieved using ImageJ software. According to the results, cell transfection using Tax plus KLF4 (mean=197±13.4) and Tax plus OCT3/4 (mean=94.7±18.0) promoted a higher UFC counting as compared to the cells trasfected with Tax (mean=21,3±8,7) or the other factors, isolately. Althouth very preliminar, the results presented here point to an interessting way for future investigations on cell transformation pathways observed in HTLV-I infection. Acknowledgements We are greatfull to Fapemig, CNPq and CAPES financial agencies for supporting the study. Keywords: HTLV, pluripotency, factors, immunology, tax Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Translational immunology and immune intervention Citation: Brito-Melo GE (2013). Influence of the factors OCT3/4, SOX2, KLF4 or c-Myc on cell transformation promoted by the oncoprotein tax from HTLV-I.. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00719 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 17 Jun 2013; Published Online: 22 Aug 2013. * Correspondence: Prof. Gustavo E Brito-Melo, Federal University of the Jequitinhonha and Mucuri Valleys - UFVM, Pharmacy, Diamantina, Not Applicable, 39100000, Brazil, gbrito1998@yahoo.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Gustavo E Brito-Melo Google Gustavo E Brito-Melo Google Scholar Gustavo E Brito-Melo PubMed Gustavo E Brito-Melo Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Differentiation of human osteosarcoma 3AB-OS stem-like cells in derivatives of the three primary germ layers as a useful &amp;lt;i&amp;gt;in vitro&amp;lt;/i&amp;gt; model to develop several purposes

A number of solid tumors contain a distinct subpopulation of cells, termed cancer stem cells (CSCs) which represent the source for tissue renewal and hold malignant potential and which would be responsible for therapy resistance. Today, the winning goal in cancer research would be to find drugs to kill both cancer cells and cancer stem cells, while sparing normal cells. Osteosarcoma is an aggressive pediatric tumor of growing bones that, despite surgery and chemotherapy, is prone to relapse. We have recently selected from human osteosarcoma MG63 cells a cancer stem-like cell line (3AB-OS), which has unlimited proliferative potential, high levels of stemness-related markers, and in vivo tumorforming capacity in xenograft assays. Here, we have shown that 3AB-OS cells can differentiate in vitro into endoderm-, mesoderm-and ectoderm-derived lineages. Cell differentiation is morphological, molecular and functional. We propose that this model system of 3AB-OS differentiation in vitro might have a number of useful purposes, among which the study of molecular mechanisms of osteosarcoma origin, and the analysis of factors involved in specification of the various cell lineages. We still do not know either what are the shared and distinguishing characters between CSCs and normal stem cells, or what is the reason why the cancer stem cells, like the normal stem cells, have the ability to differentiate toward the derivatives of the primary germ layers. It is possible that each of the differentiation capability may be exploited by CSCs to supply their needs of growing and surviving in hostile microenvironment.

Developmental signaling pathways in cancer stem cells of solid tumors

BackgroundThe intricate regulation of several signaling pathways is essential for embryonic development and adult tissue homeostasis. Cancers commonly display aberrant activity within these pathways. A population of cells identified in several cancers, termed cancer stem cells (CSCs) show similar properties to normal stem cells and evidence suggests that altered developmental signaling pathways play an important role in maintaining CSCs and thereby the tumor itself. Scope of reviewThis review will focus on the roles of the Notch, Wnt and Hedgehog pathways in the brain, breast and colon cancers. We describe the roles these pathways play in normal tissue homeostasis through the regulation of stem cell fate in these three tissues, and the experimental evidence indicating that the role of these pathways in cancers of these is directly linked to CSCs. Major conclusionsA large body of evidence is accumulating to indicate that the deregulation of Notch, Wnt and Hedgehog pathways play important roles in both normal and cancer stem cells. We are only beginning to understand how these pathways interact, how they are coordinated during normal development and adult tissue homeostasis, and how they are deregulated during cancer. However, it is becoming increasingly clear that if we are to target CSCs therapeutically, it will likely be necessary to develop combination therapies. General significanceIf CSCs are the driving force behind tumor maintenance and growth then understanding the molecular mechanisms regulating CSCs is essential. Such knowledge will contribute to better targeted therapies that could significantly enhance cancer treatments and patient survival. This article is part of a Special Issue entitled Biochemistry of Stem Cells.

Abstract CN07-02: Targeting tumor-initiating cells in medulloblastoma

Abstract The growth of many tumors has been suggested to depend on a subset of tumor cells with an extensive capacity for self-renewal, termed cancer stem cells or tumor-initiating cells (TICs). These cells are not necessarily abundant or proliferative, but because they are long-lived and often resistant to conventional therapies, they are thought to contribute to tumor resistance and recurrence. Therefore, identifying these cells and finding approaches to targeting them has become an important goal in cancer research. We recently identified a population of TICs in a mouse model of medulloblastoma, the most common malignant brain tumor in children. These cells, marked by expression of the cell surface antigen CD15, are capable of propagating tumors following transplantation, whereas CD15– cells from the same donors cannot. To gain insight into the molecular mechanisms underlying the increased tumorigenic potential of CD15+ cells, we compared their gene expression profile to that of CD15– cells. Among the genes most consistently elevated in CD15+ cells were regulators of the G2/M phases of the cell cycle. This observation led us to hypothesize that CD15+ cells display altered cell cycle regulation, and that these cells might be particularly vulnerable to inhibitors of cell cycle progression. Here we show that CD15+ cells move more quickly through the G1 and S phases of the cell cycle, and as a result contain an increased proportion of cells in G2/M. Consistent with this cell cycle skewing, CD15+ cells express higher levels of the G2/M regulators Aurora Kinase (Aurk) and Polo-like Kinase (Plk). Treatment of tumor cells with small molecule antagonists of Aurk or Plk inhibits cell cycle progression, promotes apoptosis and enhances sensitivity to other modes of chemotherapy. Treatment of tumor-bearing mice with these agents also significantly inhibits tumor progression. Importantly, cells from medulloblastoma patient-derived xenografts are also sensitive to Aurk and Plk inhibitors. These findings suggest that inhibitors of Aurk and Plk may be useful for targeting TICs and may represent a novel avenue for therapy of human medulloblastoma. Citation Format: Shirley Markant, Lourdes Adriana Esparza, Kelly Barton, Jesse Sun, Robert Wechsler-Reya,. Targeting tumor-initiating cells in medulloblastoma. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr CN07-02.

1696P - Cancer Stem Cell Markers in Clinical Pancreatic Cancer: Impact of CD44 + /CD24 + /EPCAM+ Expression on Histology and Prognosis

ABSTRACT Purpose Emerging evidence suggests that the capability of a tumor to grow and propagate is dependent on a small subset of cells within it, termed cancer stem cells (CSCs). In pancreatic cancer, CD44 + /CD24 + /EpCAM+ cells have been reported to be CSCs; however, the histological and clinical importance of these cells has not yet been investigated. Here we clarified the characteristics of CD44 + /CD24 + /EpCAM+ cells in clinical specimens of pancreatic cancer using immunohistochemical assay. Materials and methods We used surgical specimens of pancreatic ductal adenocarcinoma from 30 patients. In view of tumor heterogeneity, we randomly selected 10 high-power fields per case, and triple-positive CD44 + /CD24 + /EpCAM+ expression was identified using our scoring system. The distribution, histological characteristics, and prognostic importance of CD44 + /CD24 + /EpCAM+ cells were then analyzed. Results Among a total of 300 assessed fields, 41 (14%) were evaluated as triple-positive. The distribution of CD44 + /CD24 + /EpCAM+ cells varied widely among the 30 cases examined, and CD44 + /CD24 + /EpCAM+ expression was correlated with poor glandular differentiation and high proliferation (high Ki-67 labeling). Analysis of the three markers individually showed that CD44 and CD24 were also correlated with poor differentiation and high proliferation, while EpCAM was not. Survival analysis showed that CD44 + /CD24 + /EpCAM+ expression was not correlated with patient outcome; however, CD44 and CD24 each appeared to be correlated with poor prognosis. Conclusion In pancreatic cancer, CD44 + /CD24 + /EpCAM+ cells overlapped with poorly differentiated cells and possess high proliferative potential. In particular, double-positive CD44 + /CD24+ cells seem to have relevance when considering clinical aspects. Disclosure All authors have declared no conflicts of interest.