Subpopulation Of Stem-like Cells Research Articles

Extracellular Vesicles from High-Grade Glioma Exchange Diverse Pro-oncogenic Signals That Maintain Intratumoral Heterogeneity.

A lack of experimental models of tumor heterogeneity limits our knowledge of the complex subpopulation dynamics within the tumor ecosystem. In high-grade gliomas (HGG), distinct hierarchical cell populations arise from different glioma stem-like cell (GSC) subpopulations. Extracellular vesicles (EV) shed by cells may serve as conduits of genetic and signaling communications; however, little is known about how HGG heterogeneity may impact EV content and activity. In this study, we performed a proteomic analysis of EVs isolated from patient-derived GSC of either proneural or mesenchymal subtypes. EV signatures were heterogeneous, but reflected the molecular make-up of the GSC and consistently clustered into the two subtypes. EV-borne protein cargos transferred between proneural and mesenchymal GSC increased protumorigenic behaviors in vitro and in vivo Clinically, analyses of HGG patient data from the The Cancer Genome Atlas database revealed that proneural tumors with mesenchymal EV signatures or mesenchymal tumors with proneural EV signatures were both associated with worse outcomes, suggesting influences by the proportion of tumor cells of varying subtypes in tumors. Collectively, our findings illuminate the heterogeneity among tumor EVs and the complexity of HGG heterogeneity, which these EVs help to maintain. Cancer Res; 76(10); 2876-81. ©2016 AACR.

Stem Cells Antigen-1 Enriches for a Cancer Stem Cell-Like Subpopulation in Mouse Gastric Cancer.

There is a strong need to identify markers to enrich gastric cancer stem cells (CSCs). However, CSC enrichment markers for mouse gastric cancers have not yet been determined. In our previous study, we generated primary mouse gastric cancer cell line NCC-S1 (S1) established from a Villin-cre;Smad4(F/F) ;Trp53(F/F) ;Cdh1(F/wt) mouse and its metastatic variant cell line NCC-S1M (S1M). Interestingly, S1M cells exhibited CSC-like features, such as increased tumorigenic potential and chemoresistance. By comparing gene expression profiles between S1 and S1M cells, we identified Stem Cells Antigen-1 (Sca-1) as a cell surface marker, which was mostly upregulated in S1M. Sca-1 was upregulated in tumorspheres from S1 cells or after cisplatin treatment in S1 cells. Immunofluorescence (IF) analysis showed that approximately 7% of cancer cells exhibited positivity for Sca-1 in primary mouse gastric cancer tissues. An in vivo-limiting dilution assay showed that Sca-1(high) mouse gastric cancer cells demonstrated increased tumorigenicity compared with Sca-1(negative) cells. The Sca-1 expression was downregulated by TGF-β pathway activation and Wnt pathway inhibition in mouse gastric cancer cells. Sca-1(high) cells showed relatively low TGF-β reporter activity and high TCF/LEF1 reporter activity compared with Sca-1(negative) cells. A chromatin immunoprecipitation analysis demonstrated that Sca-1 was a β-catenin/LEF1 target gene. Sca-1(high) allografts were more resistant to cisplatin/fluorouracil chemotherapy than Sca-1(negative) allografts, and overexpressed Bcl-xL. Eighty-five mouse genes overexpressed in Sca-1(high) S1 cells compared with Sca-1(negative) cells clustered 123 pretreatment gastric cancer patient samples according to survival following chemotherapy. Taken together, Sca-1 is a novel CSC enrichment marker that mediates TGF-β and Wnt/β-catenin signaling in mouse gastric cancer. Stem Cells 2016;34:1177-1187.

Abstract BS3-2: Targeting mitochrondria with FDA-approved antibiotics may be a new therapeutic strategy to eliminate cancer stem cells

Abstract Our recent studies indicate that cancer stem cells (CSCs) have increased mitochondrial mass, which directly reflects an increase in mitochondrial biogenesis and OXPHOS capacity. Thus, we believe that more effective anti-cancer therapy would involve the strategic targeting of CSC mitochondria, to prevent or reverse tumor recurrence, metastasis and drug-resistance. Interestingly, mitochondria are originally derived from aerobic bacteria that were engulfed by eukaryotic cells and adapted over millions of years of evolution. This is known as the 'Endo-symbiotic Theory of Mitochondrial Evolution'. As a consequence, certain antibiotics target mitochondrial protein translation as a manageable side effect. We have recently proposed to harness this side effect and to re-purpose it as a therapeutic effect to target breast CSCs. In accordance with this strategy, we have already experimentally identified 4 to 5 different class of mitochondrial-targeted antibiotics that could be used halt the proliferation and eradicate breast CSCs. These antibiotics included azithromycin and doxycycline, among others. Importantly, these antibiotics showed anti-CSC activity in 12 different cell lines, across 8 different cancer types, that were originally derived from breast, DCIS, ovarian, prostate, pancreatic and lung carcinomas, as well as glioblastoma and melanoma. In this context, doxycycline appeared to be one of the most promising agents, as human clinical trials in MALT lymphoma have already shown very positive results. Similarly, clinical trials with azithromycin in lung cancer patients have also shown positive results. Thus, targeting mitochondrial protein translation, with FDA-approved antibiotics, may be a safe effective strategy for inhibiting mitochondrial function in CSCs, regardless of the tumor type or site of origin. For further information on this topic, please see the following references: Antibiotics that target mitochondria effectively eradicate cancer stem cells, across multiple tumor types: Treating cancer like an infectious disease. Lamb R, Ozsvari B, Lisanti CL, Tanowitz HB, Howell A, Martinez-Outschoorn UE, Sotgia F, Lisanti MP. Oncotarget. 2015 Mar 10;6(7):4569-84. Drug therapy: Can the mitochondrial adverse effects of antibiotics be exploited to target cancer metabolism? Killock D. Nat Rev Clin Oncol. 2015 Apr;12(4):190. High mitochondrial mass identifies a sub-population of stem-like cancer cells that are chemo-resistant. Farnie G, Sotgia F, Lisanti MP. Oncotarget. 2015 Sep 30. [Epub ahead of print] PMID: 26421710 http://www.ncbi.nlm.nih.gov/pubmed/26421710 Mitochondrial mass, a new metabolic biomarker for stem-like cancer cells: Understanding WNT/FGF-driven anabolic signaling. Lamb R, Bonuccelli G, Ozsvari B, Peiris-Pages M, Fiorillo M, Smith DL, Bevilacqua G, Mazzanti CM, McDonnell LA, Naccarato AG, Chiu M, Wynne L, Martinez-Outschoorn UE, Sotgia F, Lisanti MP. Oncotarget. 2015 Sep 28. [Epub ahead of print] PMID: 26421711 http://www.ncbi.nlm.nih.gov/pubmed/26421711 Citation Format: Lisanti MP. Targeting mitochrondria with FDA-approved antibiotics may be a new therapeutic strategy to eliminate cancer stem cells. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr BS3-2.

Identification of Stem-Like Cells in Atrial Myxoma by Markers CD44, CD19, and CD45.

Atrial myxoma is the most frequent tumor arising mainly in atrial septum and its origin remains uncertain. It has been reported that a subpopulation of stem-like cells are present in benign tumors and responsible for tumor initiation and maintenance. In this study, we investigated whether stem-like cells could contribute to the atrial cardiac myxoma. Immunohistology data confirmed that a population of cells bearing the surface markers CD19, CD45, and CD44 resided in a mucopolysaccharide-rich matrix of myxoma. Moreover, we isolated myxoma cells with phase-bright culture method and confirmed that myxoma derived cells express robust level of CD19, CD45, and CD44. Furthermore, the pluripotency of this population of cells also was validated by cardiomyocytes and smooth muscle cells differentiation in vitro. Our results indicate that primary cardiac myxoma may arise from mesenchymal stem cells with the ability to generate tumors with multilineage differentiation. In conclusion, this study for the first time verified that stem-like cells are present in atrial myxoma and this population of cells may have the capacity for myxoma initiation and progression.

Abstract B20: Targeting Olig2 co-regulators for malignant glioma therapy

Abstract The transcription factor OLIG2 is expressed within the highly tumorigenic sub-population of stem-like cells in malignant glioma and required for tumor formation in orthotopic grafts of human glioma as well as genetically defined murine glioma models. Our work (recently confirmed and extended by Suva et al., 2014) suggests that small molecule OLIG2 antagonist could serve as targeted therapy for high grade gliomas – either as a mono therapeutic or as an adjuvant to conventional radiotherapy or cytotoxic drugs. However, transcription factors in general are considered to be unattractive targets for drug development because their interactions with DNA and co-regulatory proteins involve large and complex surface area contacts. Our recent data suggests that OLIG2 interacts with distinct partner proteins based on its phosphorylation state, which in turn regulates its pro-mitogenic functions. Here we employed two independent proteomics screens (mass spectrometry and Nucleic Acid Programmable Protein Arrays, NAPPA) to identify OLIG2 co-regulators as possible surrogate targets to suppress OLIG2 function. These screens led to identification of two novel OLIG2 partner proteins: HDAC1 and proto-oncogene FYN kinase. OLIG2 is known to be a negative regulator of gene expression and histone deacetylation is required for suppression of gene expression. We demonstrate that OLIG2 functions as an HDAC1 carrier, delivering this enzyme to specific target genes (e.g., p21) and non-histone proteins like p53. HDACs are ubiquitously expressed and are known to be redundant, but we show that ablation of HDAC1 alone (using shRNA) significantly decreases the proliferation of glioma initiating cells with minimal impact on normal neural stem cells. In addition, we show that FYN kinase interacts with OLIG2 and phosphorylates it at tyrosine residues. Ablation of FYN decreases phosphorylation of OLIG2 at tyrosine residues and affects OLIG2-mediated expression of oncogenic target genes like EGFR and WNT5a. This work provides two potential co-regulators of OLIG2 which could serve as druggable surrogate targets to block OLIG2 function in malignant glioma. Further work will evaluate the efficacy of HDAC1 and FYN-specific inhibitor as single agents or as adjuvant therapy to radiation and temozolomide in orthotopic patient-derived xenograft models. Targeting factors regulating OLIG2-mediated tumor growth will have practical overtones for targeted therapies for malignant gliomas. Citation Format: Robert Kupp, Costanza Lo Cascio, Femina Rauf, Dimphna Meijer, Charles D. Stiles, Nader Sanai, Joshua LaBaer, Shwetal V. Mehta. Targeting Olig2 co-regulators for malignant glioma therapy. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr B20.

Transmembrane protein CD9 is glioblastoma biomarker, relevant for maintenance of glioblastoma stem cells.

The cancer stem cell model suggests that glioblastomas contain a subpopulation of stem-like tumor cells that reproduce themselves to sustain tumor growth. Targeting these cells thus represents a novel treatment strategy and therefore more specific markers that characterize glioblastoma stem cells need to be identified. In the present study, we performed transcriptomic analysis of glioblastoma tissues compared to normal brain tissues revealing sensible up-regulation of CD9 gene. CD9 encodes the transmembrane protein tetraspanin which is involved in tumor cell invasion, apoptosis and resistance to chemotherapy. Using the public REMBRANDT database for brain tumors, we confirmed the prognostic value of CD9, whereby a more than two fold up-regulation correlates with shorter patient survival. We validated CD9 gene and protein expression showing selective up-regulation in glioblastoma stem cells isolated from primary biopsies and in primary organotypic glioblastoma spheroids as well as in U87-MG and U373 glioblastoma cell lines. In contrast, no or low CD9 gene expression was observed in normal human astrocytes, normal brain tissue and neural stem cells. CD9 silencing in three CD133+ glioblastoma cell lines (NCH644, NCH421k and NCH660h) led to decreased cell proliferation, survival, invasion, and self-renewal ability, and altered expression of the stem-cell markers CD133, nestin and SOX2. Moreover, CD9-silenced glioblastoma stem cells showed altered activation patterns of the Akt, MapK and Stat3 signaling transducers. Orthotopic xenotransplantation of CD9-silenced glioblastoma stem cells into nude rats promoted prolonged survival. Therefore, CD9 should be further evaluated as a target for glioblastoma treatment.

IMCT-12A PHASE 1 TRIAL OF VACCINATION WITH AUTOLOGOUS DENDRITIC CELLS PULSED WITH LYSATE DERIVED FROM AN ALLOGENEIC GLIOBLASTOMA STEM-LIKE CELL LINE FOR PATIENTS WITH NEWLY DIAGNOSED OR RECURRENT GLIOBLASTOMA

BACKGROUND: Dendritic cell vaccines are a promising treatment for glioblastoma. The glioblastoma stem-like cell subpopulation is refractory to standard chemotherapy and radiation treatment, but may be susceptible to immunotherapeutic targeting. METHODS: This single-institution phase I trial is designed to assess the safety, tolerability, and potential efficacy of an autologous dendritic cell vaccine pulsed with lysate derived from a glioblastoma stem-like cell line. Patients with newly diagnosed glioblastoma (Cohort A) receive standard radiation and temozolomide in addition to vaccine. Patients with up to third glioblastoma recurrence (Cohort B) receive vaccine alone, although patients previously treated with bevacizumab are allowed to continue bevacizumab on study. Prior to enrollment, patients must undergo a qualifying gross total or near-gross total resection. Treatment consists of a Vaccine Induction Phase (weekly vaccines x 4 – for Cohort A, given upon completion of radiation), followed by a Vaccine Maintenance Phase (vaccine every 8 weeks until supply depleted). Imaging is performed during Week 4 of the Induction phase and every 8 weeks thereafter until progression. The primary objective of safety and tolerability includes adverse event grading per NCI CTC. Clinical assessment includes progression-free survival at 6 months and overall survival. Immunological testing to assess cytotoxic T-lymphocyte response will be performed at predetermined intervals. Goal enrollment is 20 patients in each cohort. Stopping rules for safety and efficacy using Bayesian sequential design will be followed. RESULTS: To date, 18 patients (7 new GBM, 11 recurrent GBM) have enrolled in this trial. No adverse events related to vaccine have been noted. CONCLUSION: This single-institution phase I dendritic cell vaccine trial builds upon our previous research efforts by specifically targeting the glioblastoma stem-like cell subpopulation that is resistant to standard radiation and chemotherapy. Accrual is proceeding rapidly, and results will be published when available.

Metformin and soybean-derived bioactive molecules attenuate the expansion of stem cell-like epithelial subpopulation and confer apoptotic sensitivity in human colon cancer cells.

Colorectal cancer (CRC) is a disease whose genesis may include metabolic dysregulation. Cancer stem cells are attractive targets for therapeutic interventions since their aberrant expansion may underlie tumor initiation, progression, and recurrence. To investigate the actions of metabolic regulators on cancer stem cell-like cells (CSC) in CRC, we determined the effects of soybean-derived bioactive molecules and the anti-diabetes drug metformin (MET), alone and together, on the growth, survival, and frequency of CSC in human HCT116 cells. Effects of MET (60μM) and soybean components genistein (Gen, 2μM), lunasin (Lun, 2μM), β-conglycinin (β-con, 3μM), and glycinin (Gly, 3μM) on HCT116 cell proliferation, apoptosis, and mRNA/protein expression and on the frequency of the CSC CD133(+)CD44(+) subpopulation by colonosphere assay and fluorescence-activated cell sorting/flow cytometry were evaluated. MET, Gen, and Lun, individually and together, inhibited HCT116 viability and colonosphere formation and, conversely, enhanced HCT116 apoptosis. Reductions in frequency of the CSC CD133(+)CD44(+) subpopulation with MET, Gen, and Lun were found to be associated with increased PTEN and reduced FASN expression. In cells under a hyperinsulinemic state mimicking metabolic dysregulation and without and with added PTEN-specific inhibitor SF1670, colonosphere formation and frequency of the CD133(+)CD44(+) subpopulation were decreased by MET, Lun and Gen, alone and when combined. Moreover, MET+Lun+Gen co-treatment increased the pro-apoptotic and CD133(+)CD44(+)-inhibitory efficacy of 5-fluorouracil under hyperinsulinemic conditions. Results identify molecular networks shared by MET and bioavailable soy food components, which potentially may be harnessed to increase drug efficacy in diabetic and non-diabetic patients with CRC.

High mitochondrial mass identifies a sub-population of stem-like cancer cells that are chemo-resistant.

Chemo-resistance is a clinical barrier to more effective anti-cancer therapy. In this context, cancer stem-like cells (CSCs) are thought to be chemo-resistant, resulting in tumor recurrence and distant metastasis. Our hypothesis is that chemo-resistance in CSCs is driven, in part, by enhanced mitochondrial function. Here, we used breast cell lines and metastatic breast cancer patient samples to begin to dissect the role of mitochondrial metabolism in conferring the CSC phenotype. More specifically, we employed fluorescent staining with MitoTracker (MT) to metabolically fractionate these cell lines into mito-high and mito-low sub-populations, by flow-cytometry. Interestingly, cells with high mitochondrial mass (mito-high) were specifically enriched in a number of known CSC markers, such as aldehyde dehydrogenase (ALDH) activity, and they were ESA+/CD24-/low and formed mammospheres with higher efficiency. Large cell size is another independent characteristic of the stem cell phenotype; here, we observed a >2-fold increase in mitochondrial mass in large cells (>12-μm), relative to the smaller cell population (4-8-μm). Moreover, the mito-high cell population showed a 2.4-fold enrichment in tumor-initiating cell activity, based on limiting dilution assays in murine xenografts. Importantly, primary human breast CSCs isolated from patients with metastatic breast cancer or a patient derived xenograft (PDX) also showed the co-enrichment of ALDH activity and mitochondrial mass. Most significantly, our investigations demonstrated that mito-high cells were resistant to paclitaxel, resulting in little or no DNA damage, as measured using the comet assay. In summary, increased mitochondrial mass in a sub-population of breast cancer cells confers a stem-like phenotype and chemo-resistance. As such, our current findings have important clinical implications for over-coming drug resistance, by therapeutically targeting the mito-high CSC population.

Effect of Emodin Combined with AZT on the Proliferation and the Expression of BCL-2, NF-κB, TGF-β in the Leukemia Stem Cells-KG-1a cells

To investigate the effect of Emodin combined with 3'-azido-3'-deoxythymidine (AZT) on the proliferation and apoptosis of concentrated leukemia stem cells (CLSC)-human acute myeloid leukemia KG-la cells and expression of BCL-2, NF-κB and TGF-β. The tumor stem cell-like subpopulation in human leukemia cell line KG-1a was enriched with 5-fluorouracil (5-FU). The CD34⁺ CD38⁻ subpopulation in the KG-1a cells was detected with flow cytometry, the cell proliferation was detected by MTT method to study the of Emodin and AZT in the CLSC. The cell apoptosis was analyzed by flow cytometry. The expression of NF-κB, BCL-2 and TGF-β mRNA and proteins were measured with RT-PCR and Western blot respectively. As compared with cells treated with mentioned above drugs alone, the inhibition of proliferation potential and apoptosis rate of cells in combination group markedly increase with time and concentration dependent member (P < 0.01), the expression of NF-κB, BCL-2 and TGF-β mRNA and proteins decreased. Emodin combined AZT can synergistically inhibit the proliferation, induce cell apoptosis, and down regulate the expression of NF-κB, BCL-2 and TGF-β mRNA and proteins in the CLSC, the possible mechanism of synergistic effect may be associated with inhibiton of BCL-2 activation and down-regulation of the expression of NF-κB, and TGF-β.

Abstract 5468: Tirapazamine as a strategy to prevent cell dissemination and overcome drug resistance

Abstract Introduction: Multiple myeloma (MM) and Waldenström's macroglobulinemia (WM) are lymphoplasmacytic malignancies which are characterized by continuous spread through cell trafficking in and out of the BM. We have recently shown that the spread process in these diseases is mediated by hypoxia. Moreover, despite the introduction of novel therapies, cancer patients relapse due to the development of a drug-resistant stem cell-like subpopulation, which we recently found to be, at least in part, promoted by hypoxia. Therefore, in this study we aimed to overcome spread and drug resistance in MM and WM by inhibition of the hypoxic responses in these cells. Tirapazamine (TPZ) is a hypoxia-activated pro-drug causing cell apoptosis, which has been shown to improve cancer patient outcome when combined with radiotherapy. We use TPZ for the first time in hematologic malignancies to target the drug-resistant cancer cells and sensitize them to therapy. Procedures: Cell survival was tested on MM (MM1.s, H929, OPM1, RPMI8226) and WM (BCWM.1, MWCL.1) cell lines exposed to normoxia (21% O2) or hypoxia (1% O2) for 24hrs in the presence of bortezomib and carfilzomib, different concentrations of TPZ (in order to obtain an IC50), or combination of those drugs using MTT assay. For adhesion assay, cancer cells were pre-labeled with calcein-AM, treated with TPZ, applied to unlabeled endothelial cells, non-adherent cells were washed after 1hr, and adherent cells were analyzed by a fluorescent reader. Chemotactic properties of cells were tested using a transwell chamber and the migrated cells were counted by flow cytometry. For in vivo study, 5 × 106 MM1s-Luc-GFP cells were injected i.v. into 6 SCID mice and tumor progression was monitored for 3 weeks by bioluminescent imaging. All mice were treated with (1) high-dose bortezomib (1.5mg/kg) only (n = 3), or (2) bortezomib and TPZ (40mg/kg; n = 3), all administered i.p. sequentially twice a week. Bone marrow and peripheral blood were collected and analyzed for the presence of GFP+ cells by flow cytometry. Summary of data: We found that hypoxia induced drug resistance to bortezomib and carfilzomib of MM and WM cells, and that TPZ was active in a dose-dependent manner only in hypoxic conditions. Combination of TPZ with bortezomib and carfilzomib, resensitized cancer cells to death in hypoxia. Addition of TPZ increased adhesion of cancer cells to endothelial cells, and decreased chemotactic properties of cancer cells in hypoxia in vitro. Moreover, post-treatment residual cancer cells were further decreased using TPZ, preventing recurrence of MM in vivo. Conclusion: We report that TPZ prevents hypoxia-induced metastasis and overcomes hypoxia-induced drug resistance in MM and WM. This is the first study to show the efficacy of TPZ in hematologic malignancies in general and in MM and WM in particular. This data provide a preclinical basis for future clinical trials testing efficacy of TPZ in MM and WM. Citation Format: Barbara Muz, Pilar de la Puente, Feda Azab, Micah Luderer, Abdel Kareem Azab. Tirapazamine as a strategy to prevent cell dissemination and overcome drug resistance. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5468. doi:10.1158/1538-7445.AM2015-5468

Targeting Aggressive Cancer Stem Cells in Glioblastoma.

Glioblastoma (GBM) is the most common and fatal type of primary brain tumor. Gliosarcoma (GSM) is a rarer and more aggressive variant of GBM that has recently been considered a potentially different disease. Current clinical treatment for both GBM and GSM includes maximal surgical resection followed by post-operative radiotherapy and concomitant and adjuvant chemotherapy. Despite recent advances in treating other solid tumors, treatment for GBM and GSM still remains palliative, with a very poor prognosis and a median survival rate of 12–15 months. Treatment failure is a result of a number of causes, including resistance to radiotherapy and chemotherapy. Recent research has applied the cancer stem cells theory of carcinogenesis to these tumors, suggesting the existence of a small subpopulation of glioma stem-like cells (GSCs) within these tumors. GSCs are thought to contribute to tumor progression, treatment resistance, and tumor recapitulation post-treatment and have become the focus of novel therapy strategies. Their isolation and investigation suggest that GSCs share critical signaling pathways with normal embryonic and somatic stem cells, but with distinct alterations. Research must focus on identifying these variations as they may present novel therapeutic targets. Targeting pluripotency transcription factors, SOX2, OCT4, and Nanog homeobox, demonstrates promising therapeutic potential that if applied in isolation or together with current treatments may improve overall survival, reduce tumor relapse, and achieve a cure for these patients.

Transforming growth factor-beta1 promotes the migration and invasion of sphere-forming stem-like cell subpopulations in esophageal cancer

Esophageal cancer is one of the most lethal solid malignancies. Mounting evidence demonstrates that cancer stem cells (CSCs) are able to cause tumor initiation, metastasis and responsible for chemotherapy and radiotherapy failures. As CSCs are thought to be the main reason of therapeutic failure, these cells must be effectively targeted to elicit long-lasting therapeutic responses. We aimed to enrich and identify the esophageal cancer cell subpopulation with stem-like properties and help to develop new target therapy strategies for CSCs. Here, we found esophageal cancer cells KYSE70 and TE1 could form spheres in ultra low attachment surface culture and be serially passaged. Sphere-forming cells could redifferentiate and acquire morphology comparable to parental cells, when return to adherent culture. The sphere-forming cells possessed the key criteria that define CSCs: persistent self-renewal, overexpression of stemness genes (SOX2, ALDH1A1 and KLF4), reduced expression of differentiation marker CK4, chemoresistance, strong invasion and enhanced tumorigenic potential. SB525334, transforming growth factor-beta 1(TGF-β1) inhibitor, significantly inhibited migration and invasion of sphere-forming stem-like cells and had no effect on sphere-forming ability. In conclusion, esophageal cancer sphere-forming cells from KYSE70 and TE1 cultured in ultra low attachment surface possess cancer stem cell properties, providing a model for CSCs targeted therapy. TGF-β1 promotes the migration and invasion of sphere-forming stem-like cells, which may guide future studies on therapeutic strategies targeting these cells.

A CDC20-APC/SOX2 Signaling Axis Regulates Human Glioblastoma Stem-like Cells.

Glioblastoma harbors a dynamic subpopulation of glioblastoma stem-like cells (GSCs) that can propagate tumors in vivo and is resistant to standard chemoradiation. Identification of the cell-intrinsic mechanisms governing this clinically important cell state may lead to the discovery of therapeutic strategies for this challenging malignancy. Here, we demonstrate that the mitotic E3 ubiquitin ligase CDC20-anaphase-promoting complex (CDC20-APC) drives invasiveness and self-renewal in patient tumor-derived GSCs. Moreover, CDC20 knockdown inhibited and CDC20 overexpression increased the ability of human GSCs to generate brain tumors in an orthotopic xenograft model in vivo. CDC20-APC control of GSC invasion and self-renewal operates through pluripotency-related transcription factor SOX2. Our results identify a CDC20-APC/SOX2 signaling axis that controls key biological properties of GSCs, with implications for CDC20-APC-targeted strategies in the treatment of glioblastoma.

CD15 Expression Does Not Identify a Phenotypically or Genetically Distinct Glioblastoma Population.

: Recent research has focused on the hypothesis that the growth and regeneration of glioblastoma (GB) is sustained by a subpopulation of self-renewing stem-like cells. This has led to the prediction that molecular markers for cancer stem cells in GB may provide a treatment target. One candidate marker is CD15: we wanted to determine if CD15 represented a credible stem cell marker in GB. We first demonstrated that CD15-positive (CD15+) cells were less proliferative than their CD15-negative (CD15-) counterparts in 10 patient GB tumors. Next we compared the proliferative activity of CD15+ and CD15- cells in vitro using tumor-initiating primary GB cell lines (TICs) and found no difference in proliferative behavior. Furthermore, TICs sorted for CD15+ and CD15- were not significantly different cytogenetically or in terms of gene expression profile. Sorted single CD15+ and CD15- cells were equally capable of reconstituting a heterogeneous population containing both CD15+ and CD15- cells over time, and both CD15+ and CD15- cells were able to generate tumors in vivo. No difference was found in the phenotypic or genomic behavior of CD15+ cells compared with CD15- cells from the same patient. Moreover, we found that in vitro, cells were able to interconvert between the CD15+ and CD15- states. Our data challenge the utility of CD15 as a cancer stem cell marker. The data from this study contribute to the ongoing debate about the role of cancer stem cells in gliomagenesis. Results showed that CD15, a marker previously thought to be a cancer stem-like marker in glioblastoma, could not isolate a phenotypically or genetically distinct population. Moreover, isolated CD15-positive and -negative cells were able to generate mixed populations of glioblastoma cells in vitro.

Normal vs cancer thyroid stem cells: the road to transformation.

Recent investigations in thyroid carcinogenesis have led to the isolation and characterisation of a subpopulation of stem-like cells, responsible for tumour initiation, progression and metastasis. Nevertheless, the cellular origin of thyroid cancer stem cells (SCs) remains unknown and it is still necessary to define the process and the target population that sustain malignant transformation of tissue-resident SCs or the reprogramming of a more differentiated cell. Here, we will critically discuss new insights into thyroid SCs as a potential source of cancer formation in light of the available information on the oncogenic role of genetic modifications that occur during thyroid cancer development. Understanding the fine mechanisms that regulate tumour transformation may provide new ground for clinical intervention in terms of prevention, diagnosis and therapy.

Radiotherapy followed by aurora kinase inhibition targets tumor-propagating cells in human glioblastoma.

Glioblastoma (GBM) is the most common malignant primary brain tumor. Radiotherapy fails to eliminate subpopulations of stem-like tumor-propagating cells (TPC), resulting in tumor regrowth. To identify kinases that promote TPC self-renewal rather than increasing proliferation in human GBM cultures, we screened a library of 54 nonselective tool compounds and determined their kinase inhibitor profiles in vitro. Most compounds inhibited aurora kinase (AURK) activity and blocked TPC self-renewal, while inducing GBM cell polynucleation and apoptosis. To prevent regrowth by TPCs, we used a priming dose of radiation followed by incubation with the pan-AURK inhibitor VX680 to block self-renewal and induce apoptosis in GBM cultures. In mice xenografted with human GBM cells, radiotherapy followed by VX680 treatment resulted in reduced tumor growth and increased survival relative to either monotherapy alone or VX680 treatment before radiation. Our results indicate that AURK inhibition, subsequent to radiation, may enhance the efficacy of radiotherapy by targeting radioresistant TPCs in human GBMs.

Inositol Polyphosphate-5-Phosphatase F (INPP5F) inhibits STAT3 activity and suppresses gliomas tumorigenicity

Glioblastoma (GBM), the most common type of primary malignant brain tumors harboring a subpopulation of stem-like cells (GSCs), is a fast-growing and often fatal tumor. Signal Transducer and Activator of Transcription 3 (STAT3) is one of the major signaling pathways in GSCs maintenance but the molecular mechanisms underlying STAT3 deregulation in GSCs are poorly defined. Here, we demonstrate that Inositol Polyphosphate-5-Phosphatase F (INPP5F), one of the polyphosphoinositide phosphatases, is differentially expressed in GSCs from glioma patients, and is identified as an inhibitor of STAT3 signaling via interaction with STAT3 and inhibition of its phosphorylation. Constitutively expressed INPP5F showed to suppress self-renewal and proliferation potentials of glioblastoma cells and reduced tumorigenicity of glioblastoma. In addition, loss of INPP5F gene in gliomas is significantly correlated with lower overall patient survivals. These findings suggest that INPP5F is a potential tumor suppressor in gliomas via inhibition of STAT3 pathway, and that deregulation of INPP5F may lead to contribution to gliomagenesis.

Coordinate hyperactivation of Notch1 and Ras/MAPK pathways correlates with poor patient survival: novel therapeutic strategy for aggressive breast cancers.

Aberrant activation of Notch and Ras pathways has been detected in breast cancers. A synergy between these two pathways has also been shown in breast cell transformation in culture. Yet, the clinical relevance of Notch-Ras cooperation in breast cancer progression remains unexplored. In this study, we show that coordinate hyperactivation of Notch1 and Ras/MAPK pathways in breast cancer patient specimens, as assessed by IHC for cleaved Notch1 and pErk1/2, respectively, correlated with early relapse to vital organs and poor overall survival. Interestingly, majority of such Notch1(high)Erk(high) cases encompassed the highly aggressive triple-negative breast cancers (TNBC), and were enriched in stem cell markers. We further show that combinatorial inhibition of Notch1 and Ras/MAPK pathways, using a novel mAb against Notch1 and a MEK inhibitor, respectively, led to a significant reduction in proliferation and survival of breast cancer cells compared with individual inhibition. Combined inhibition also abrogated sphere-forming potential, and depleted the putative cancer stem-like cell subpopulation. Most importantly, combinatorial inhibition of Notch1 and Ras/MAPK pathways completely blocked tumor growth in a panel of breast cancer xenografts, including the TNBCs. Thus, our study identifies coordinate hyperactivation of Notch1 and Ras/MAPK pathways as novel biomarkers for poor breast cancer outcome. Furthermore, based on our preclinical data, we propose combinatorial targeting of these two pathways as a treatment strategy for highly aggressive breast cancers, particularly the TNBCs that currently lack any targeted therapeutic module.

OP34 * TARGETING RAD51 TO RADIOSENSITISE GLIOBLASTOMA STEM CELLS

INTRODUCTION: The capacity of a sub-population of stem-like cells (Glioma stem cells, GSC) to withstand treatment is thought to play a major role in tumour recurrence. It is becoming increasingly apparent that these GSC express elevated levels of DNA repair proteins including the homologous recombination protein Rad51. We have investigated whether targeting Rad51 is a viable mehod of radio-sensitising GSC. METHOD: Patient derived GSC were characterised for repair protein levels using western blot and IF microscopy when cells were cultured in stem cell permissive media and in differentiating conditions (exposure to BMP-4 and/or serum). The expression of Rad51 and its co-expression with stem cell markers in tumour samples was determined using IHC. We used 2 pharmacological inhibitors of Rad51 (RI-1 and B02) on undifferentiated cells to investigate the effect of inhibition on sensitivity to clinically relevant radiation doses RESULTS: Levels of Rad51 were elevated in GSC compared to normal human astrocytes. Differentiation of these cells reduces the expression of Rad51 in parallel with the stem cell markers SOX2 and Nestin. Rad51 was also found to preferentially co-localise with Nestin in tumour samples. The Rad51 inhibitors reduced Rad51 foci formation following irradiation in GSC without affecting γH2AX foci. Both inhibitors caused radiosensitisation when combined with single radiation doses between 1 and 5 Gy, with dose modifying factors of between 1.33 and 1.46 CONCLUSION: The increased expression of Rad51 in GSC and the ability of Rad51 inhibitors to radiosensitise these cells suggest that Rad51 may be a specific target for GSC and that targeting this repair pathway may lead to improved response to radiotherapy in GBM.