CSC-like Subpopulations Research Articles

Exosomal-microRNA transcriptome profiling of Parental and CSC-like MDA-MB-231 cells in response to cisplatin treatment

Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype with higher risk of metastasis and cancer reoccurrence. Cisplatin is one of the potential anticancer drugs for treating TNBC, where its effectiveness remains challenged by frequent occurrence of cisplatin resistance. Since acquirement of drug resistance often being associated with presence of cancer stem cells (CSCs), investigation has been conducted, suggesting CSC-like subpopulation to be more resistant to cisplatin than their parental counterpart. On the other hand, plethora evidences showed the transmission of exosomal-miRNAs are capable of promoting drug resistance in breast cancers. In this study, we aim to elucidate the differential expression of exosomal-microRNAs profile and reveal the potential target genes in correlation to cisplatin resistance associated with CSC-like subpopulation by using TNBC cell line (MDA-MB-231). Utilizing next generation sequencing and Nanostring techniques, cisplatin-induced dysregulation of exosomal-miRNAs were evaluated in maximal for CSC-like subpopulation as compared to parental cells. Intriguingly, more oncogenic exosomal-miRNAs profile was detected from treated CSC-like subpopulation, which may correlate to enhancement of drug resistance and maintenance of CSCs. In treated CSC-like subpopulation, unique clusters of exosomal-miRNAs namely miR-221–3p, miR-196a-5p, miR-17–5p and miR-126–3p were predicted to target on six genes (ATXN1, LATS1, GSK3β, ITGA6, JAG1 and MYC), aligned with previous finding which demonstrated dysregulation of these genes in treated CSC-like subpopulation. Our results highlight the potential correlation of exosomal-miRNAs and their target genes as well as novel perspectives of the corresponding pathways that may be essential to contribute to the attenuated cytotoxicity of cisplatin in CSC-like subpopulation.

Small cell lung cancer stem cells display mesenchymal properties and exploit immune checkpoint pathways in activated cytotoxic T lymphocytes

Small cell lung cancer (SCLC) is an aggressive tumor type with early dissemination and distant metastasis capacity. Even though optimal chemotherapy responses are observed initially in many patients, therapy resistance is almost inevitable. Accordingly, SCLC has been regarded as an archetype for cancer stem cell (CSC) dynamics. To determine the immune-modulatory influence of CSC in SCLC, this study focused on the characterization of CD44+CD90+ CSC-like subpopulations in SCLC. These cells displayed mesenchymal properties, differentiated into different lineages and further contributed to CD8+ cytotoxic T lymphocytes (CTL) responses. The interaction between CD44+CD90+ CSC-like cells and T cells led to the upregulation of checkpoint molecules PD-1, CTLA-4, TIM-3, and LAG3. In the patient-derived lymph nodes, CD44+ SCLC metastases were also observed with T cells expressing PD-1, TIM-3, or LAG3. Proliferation and IFN-γ expression capacity of TIM-3 and LAG3 co-expressing CTLs are adversely affected over long-time co-culture with CD44+CD90+ CSC-like cells. Moreover, especially through IFN-γ secreted by the T cells, the CSC-like SCLC cells highly expressed PD-L1 and PD-L2. Upon a second encounter with immune-experienced, IFN-γ-stimulated CSC-like SCLC cells, both cytotoxic and proliferation capacities of T cells were hampered. In conclusion, our data provide evidence for the superior potential of the SCLC cells with stem-like and mesenchymal properties to gain immune regulatory capacities and cope with cytotoxic T cell responses. With their high metastatic and immune-modulatory assets, the CSC subpopulation in SCLC may serve as a preferential target for checkpoint blockade immunotherapy.

Isolation and Characterization of Human Colon Adenocarcinoma Stem-Like Cells Based on the Endogenous Expression of the Stem Markers.

Background: Cancer stem cells’ (CSCs) self-maintenance is regulated via the pluripotency pathways promoting the most aggressive tumor phenotype. This study aimed to use the activity of these pathways for the CSCs’ subpopulation enrichment and separating cells characterized by the OCT4 and SOX2 expression. Methods: To select and analyze CSCs, we used the SORE6x lentiviral reporter plasmid for viral transduction of colon adenocarcinoma cells. Additionally, we assessed cell chemoresistance, clonogenic, invasive and migratory activity and the data of mRNA-seq and intrinsic disorder predisposition protein analysis (IDPPA). Results: We obtained the line of CSC-like cells selected on the basis of the expression of the OCT4 and SOX2 stem cell factors. The enriched CSC-like subpopulation had increased chemoresistance as well as clonogenic and migration activities. The bioinformatic analysis of mRNA seq data identified the up-regulation of pluripotency, development, drug resistance and phototransduction pathways, and the downregulation of pathways related to proliferation, cell cycle, aging, and differentiation. IDPPA indicated that CSC-like cells are predisposed to increased intrinsic protein disorder. Conclusion: The use of the SORE6x reporter construct for CSCs enrichment allows us to obtain CSC-like population that can be used as a model to search for the new prognostic factors and potential therapeutic targets for colon cancer treatment.

CD117/c-kit defines a prostate CSC-like subpopulation driving progression and TKI resistance

Cancer stem-like cells (CSCs) are associated with cancer progression, metastasis, and recurrence, and may also represent a subset of circulating tumor cells (CTCs). In our prior study, CTCs in advanced prostate cancer patients were found to express CD117/c-kit in a liquid biopsy. Whether CD117 expression played an active or passive role in the aggressiveness and migration of these CTCs remained an open question. In this study, we show that CD117 expression in prostate cancer patients is associated with decreased overall and progression-free survival and that activation and phosphorylation of CD117 increases in prostate cancer patients with higher Gleason grades. To determine how CD117 expression and activation by its ligand stem cell factor (SCF, kit ligand, steel factor) alter prostate cancer aggressiveness, we used C4-2 and PC3-mm human prostate cancer cells, which contain a CD117+ subpopulation. We demonstrate that CD117+ cells display increased proliferation and migration. In prostaspheres, CD117 expression enhances sphere formation. In both 2D and 3D cultures, stemness marker gene expression is higher in CD117+ cells. Using xenograft limiting dilution assays and serial tumor initiation assays, we show that CD117+ cells represent a CSC population. Combined, these data indicate that CD117 expression potentially promotes tumor initiation and metastasis. Further, in cell lines, CD117 activation by SCF promotes faster proliferation and invasiveness, while blocking CD117 activation with tyrosine kinase inhibitors (TKIs) decreased progression in a context-dependent manner. We demonstrate that CD117 expression and activation drives prostate cancer aggressiveness through the CSC phenotype and TKI resistance.

Lipid desaturation-associated endoplasmic reticulum stress regulates MYCN gene expression in hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths worldwide due to its high rate of recurrence, in part because of cancer stem cell (CSC)-dependent “field cancerization”. Recently, we identified that the oncogene v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN) marked CSC-like subpopulations in heterogeneous HCC and served as a therapeutic target and prognostic marker for HCC. In this study, we explored the molecular basis of upregulated MYCN gene expression in HCC cells. Liquid chromatograph time-of-flight mass spectrometry-based metabolome analysis demonstrated that the content of unsaturated fatty acids was increased in MYCN high expression (MYCNhigh) CSC-like HCC cells. Inhibition of lipid desaturation using either the chemical inhibitor or siRNA/shRNA against stearoyl-CoA desaturase-1 (SCD1) suppressed cell proliferation as well as MYCN gene expression in MYCNhigh HCC cells, grown as both monolayer and spheres. Further mechanistic study using RNA-seq based transcriptome analysis revealed that endoplasmic reticulum (ER) stress related signaling networks such as endocannabinoid cancer inhibition pathway were under the control of SCD1 in MYCNhigh HCC cells. Furthermore, the expression of ER stress-inducible transcription suppressor cyclic AMP-dependent transcription factor (ATF3) was downregulated in MYCNhigh CSC-like HCC cells and CSC-rich spheroids, which was upregulated by inhibition of lipid desaturation or treatment with acyclic retinoid (ACR). Lipid profiling using NMR spectroscopy revealed that the ACR dramatically reduced the content of unsaturated fatty acids in HCC cells. The chemical inducer of ER stress inhibited MYCN gene expression, while the chemical inhibitor of ER stress or knockdown of ATF3 gene expression partially rescued the suppression of MYCN gene expression by ACR in MYCNhigh HCC cells. These data suggested that lipid desaturation-mediated ER stress signaling regulates MYCN gene expression in HCC cells and serves as a promising therapeutic target for the treatment and prevention of HCC.

Discovery, characterization and potential roles of a novel NF-YAx splice variant in human neuroblastoma

BackgroundIdentification of novel cancer-associated splice variants is of potential diagnostic, prognostic and therapeutic importance. NF-Y transcription factor is comprised of NF-YA, NF-YB and NF-YC subunits, binds inverted CCAAT-boxes in ≈70% of gene promoters, regulates > 1000 cancer-associated genes and proteins involved in proliferation, staminality, differentiation, apoptosis, metabolism and is subject to component alternative splicing. RT-PCR evaluation of alternative NF-YA splicing in primary human neuroblastomas (NBs), led to discovery of a novel NF-YAx splice variant, also expressed during mouse embryo development and induced by doxorubicin in NB cells. Here, we report the discovery and characterisation of NF-YAx and discus its potential roles in NB.MethodsNF-YAx cDNA was RT-PCR-cloned from a stage 3 NB (provided by the Italian Association of Haematology and Paediatric Oncology, Genova, IT), sequenced and expressed as a protein using standard methods and compared to known fully-spliced NF-YAl and exon B-skipped NF-YAs isoforms in: EMSAs for capacity to form NF-Y complexes; by co-transfection, co-immunoprecipitation and Western blotting for capacity to bind Sp1; by IF for localisation; in AO/EtBr cell-death and colony formation assays for relative cytotoxicity, and by siRNA knockdown, use of inhibitors and Western blotting for potential mechanisms of action. Stable SH-SY5Y transfectants of all three NF-YA isoforms were also propagated and compared by RT-PCR and Western blotting for differences in cell-death and stem cell (SC)-associated gene expression, in cell-death assays for sensitivity to doxorubicin and in in vitro proliferation, substrate-independent growth and in vivo tumour xenograft assays for differences in growth and tumourigenic capacity.ResultsNF-YAx was characterized as a novel variant with NF-YA exons B, D and partial F skipping, detected in 20% of NF-YA positive NBs, was the exclusive isoform in a stage 3 NB, expressed in mouse stage E11.5–14 embryos and induced by doxorubicin in SH-SY5Y NB cells. The NF-YAx protein exhibited nuclear localisation, competed with other isoforms in CCAAT box-binding NF-Y complexes but, in contrast to other isoforms, did not bind Sp1. NF-YAx expression in neural-related progenitor and NB cells repressed Bmi1 expression, induced KIF1Bβ expression and promoted KIF1Bβ-dependent necroptosis but in NB cells also selected tumourigenic, doxorubicin-resistant, CSC-like sub-populations, resistant to NF-YAx cytotoxicity.ConclusionsThe discovery of NF-YAx in NBs, its expression in mouse embryos and induction by doxorubicin in NB cells, unveils a novel NF-YA splice mechanism and variant, regulated by and involved in development, genotoxic-stress and NB. NF-YAx substitution of other isoforms in NF-Y complexes and loss of capacity to bind Sp1, characterises this novel isoform as a functional modifier of NF-Y and its promotion of KIF1Bβ-dependent neural-lineage progenitor and NB cell necroptosis, association with doxorubicin-induced necroptosis and expression in mouse embryos coinciding with KIF1Bβ-dependent sympathetic neuroblast-culling, confirm a cytotoxic function and potential role in suppressing NB initiation. On the other hand, the in vitro selection of CSC-like NB subpopulations resistant to NF-YAx cytotoxicity not only helps to explain high-level exclusive NF-YAx expression in a stage 3 NB but also supports a role for NF-YAx in disease progression and identifies a potential doxorubicin-inducible mechanism for post-therapeutic relapse.

A NF-ĸB-Activin A signaling axis enhances prostate cancer metastasis.

Metastasis is a main cause of death in prostate cancer (PCa). To dissect the molecular cues from cancer cell-microenvironment interaction that drive metastatic cascade, bone metastatic PCa cells were intravenously implanted into zebrafish embryos and mice tibia forming metastatic lesions. Transcriptomic analysis showed an elevated expression of stemness genes, pro-inflammatory cytokines and TGF-β family member Activin A in the cancer cells at metastatic onset in both animal models. Consistently, analysis of clinical datasets revealed that the expression of Activin A is specifically elevated in metastases and correlates with poor prognosis in stratified high-risk PCa patients. It is further unveiled that the microenvironment induced Activin A expression by NF-κB activation. The elevated level of Activin A enhanced the invasive ALDHhi CSC-like phenotypes and PCa proliferation by activation of Smad and ERK1/2 signaling driving metastasis. Suppression of Activin A or Activin receptor significantly reduced the CSC-like subpopulation, invasion, metastatic growth, and bone lesion formation in zebrafish and mice xenografts, suggesting a functional role of NF-κB-dependent Activin A in PCa metastasis. Overall, our study demonstrates that human PCa cells can display a comparable response with the microenvironment in zebrafish and mice xenografts. Combining both animal models, we uncovered the microenvironment-dependent activin signaling as an essential driver in PCa metastasis with therapeutic potential.

Highly versatile cancer photoimmunotherapy using photosensitizer-conjugated avidin and biotin-conjugated targeting antibodies

BackgroundPhotoimmunotherapy (PIT) employing antibody-photosensitizer conjugates is a promising treatment for cancer. However, the fixed antigen specificity severely limits the efficacy and the applicability. Here we describe a universal strategy for PIT of cancer by using a near-infrared (NIR) photosensitizer IRDye700DX-conjugated NeutrAvidin, designated as AvIR, together with various biotinylated antibodies (BioAbs) for cellular targeting.MethodsCytotoxicity of AvIR-mediated PIT was evaluated by fluorescence imaging and cell viability assay. Phototoxic effect on tumorigenicity was assessed by tumorsphere-formation assay and Matrigel invasion assay. Cancer stem cell-like side-population (SP) cells were identified by flow cytometry.ResultsCHO cells stably expressing carcinoembryonic antigen or EpCAM were pre-labeled with each BioAb for the corresponding antigen, followed by AvIR administration. NIR light irradiation specifically killed the targeted cells, but not off-targets, demonstrating that the AvIR-mediated PIT does work as expected. CSC-like subpopulation of MCF-7 cells (CD24low/CD44high) and SP of HuH-7 cells (CD133+/EpCAM+) were effectively targeted and photokilled by AvIR-PIT with anti-CD44 BioAb or anti-CD133/anti-EpCAM BioAbs, respectively. As results, the neoplastic features of the cell lines were sufficiently suppressed. Cancer-associated fibroblast (CAF)-targeted AvIR-PIT by using anti-fibroblast activation protein BioAb showed an abolishment of CAF-enhanced clonogenicity of MCF-7 cells.ConclusionsCollectively, our results demonstrate that AvIR-mediated PIT can greatly broaden the applicable range of target specificity, with feasibility of efficacious and integrative control of CSC and its microenvironment.

Abstract B21: Epigenetic modifiers MLL1 and JMJD3 regulate neuroblastoma tumorigenicity by maintaining a cancer stem cell-like population

Abstract High-risk neuroblastoma (NB) represents a major clinical challenge in pediatric oncology. Despite significant dose escalation of intense therapies, long-term survival for NB patients remains poor (<45%), and the disease accounts for almost 15% of all pediatric cancer deaths. Relapse of metastatic, drug-resistant disease and treatment-related toxicities mandates the development of new therapeutic strategies. Recently, we discovered a highly tumorigenic, chemoresistant, and self-renewing subpopulation in NB with features similar to cancer stem cells (CSCs). This G-CSF receptor (CD114)-expressing subpopulation can escape initial therapy and cause aggressively invasive relapsed disease. Therefore, developing direct targeting strategies and understanding for the molecular mechanisms maintaining this NB CSC-like subpopulation is essential to delineate effective therapeutics for NB patients. By using siRNA screening approach and low-density pathway arrays, we found that the epigenetic regulators mixed-lineage leukemia-1 (MLL1; KMT2A; a H3K4me3 methyltransferase) and Jumonji D3 (JMJD3; KDM6B; a H3K27me3 demethylase) are important epigenetic regulators for NB proliferation and growth. Interestingly, we found that epigenetic regulators MLL1 and JMJD3 are aberrantly expressed in CD114+ subpopulation and regulate the expression of the G-CSF receptor gene (CSF3R) itself, by maintaining active histone modifications at the promoter locus. Inhibition of MLL1 and JMJD3 with specific small-molecule inhibitors reverses the histone patterns at CSF3R promoter and blocks gene expression, induces apoptosis selectively in CD114+ cells, and inhibits overall NB proliferation in vitro. Furthermore, inhibiting MLL1 and JMJD3 leads to dramatic tumor regression (p<0.001) and reduction in incidences of metastasis (p<0.001) in vivo. Most interestingly, both of these inhibitors increase the overall survival of the treated mice in vivo. As expected, reduction in tumor size was significantly correlated with the reduction in tumor CD114+ cells. Taken together, these data highlight that: a) neuroblastoma is maintained by the complex interplay of epigenetic modifiers, and b) direct targeting of these epigenetic modifiers and combining this approach with current therapy is a novel therapeutic approach to high-risk NB. Citation Format: Saurabh Agarwal, Julie Tomolonis, Sanjeev Vasudevan, Jason Shohet. Epigenetic modifiers MLL1 and JMJD3 regulate neuroblastoma tumorigenicity by maintaining a cancer stem cell-like population [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B21.

Reactive oxygen species mediate cancer stem-like cells and determine bortezomib sensitivity via Mcl-1 and Zeb-1 in mantle cell lymphoma

Mantle cell lymphoma (MCL) is an aggressive, incurable non-Hodgkin B-cell lymphoma with good initial response to therapy then subsequently relapse. Cancer stem cells (CSCs) are considered to be an underlying cause of these inevitable drug resistance and tumor regrowth, but how CSCs are regulated is largely unknown. We demonstrate here for the first time the existence of CSC-like subpopulations that are modulated by reactive oxygen species (ROS) in MCL cell lines and patient-derived primary cells in an inverse correlation with bortezomib (BTZ) sensitivity. Using various known donors and inhibitors of cellular superoxide (O2−), hydrogen peroxide (H2O2) and hydroxyl radical (OH), we unveil their distinct roles in the regulation of CSC-like subpopulations and thus MCL response to BTZ. O2− inhibits CSC-like cells and sensitizes BTZ-induced apoptosis, whereas H2O2 conversely enriches CSC-like cells and protects against apoptosis and OH has minimal effects. We further observed that an anti-apoptotic Mcl-1 and a transcription factor Zeb-1 are favorable targets of O2− and H2O2, respectively. Using small molecule inhibition, ectopic expression and CRISPR/Cas9-mediated gene manipulation, we verified the roles of Mcl-1 and Zeb-1 in CSC and apoptosis regulation by O2− and H2O2. Our findings provide a novel mechanistic insight into the significance of redox status of MCL cells in determining their drug response via CSC-like subpopulations, which are imperative to a better understanding of therapeutic resistance and relapse.

Abstract 4756: A SPP1-EGFR pathway links stem-like properties of KRAS-mutated lung cancer to radiation resistance

Abstract Lung cancers with activating mutations in the KRAS oncogene present a major clinical challenge due to poor prognosis and frequent treatment resistance, particularly to agents targeting the epidermal growth factor receptor (EGFR). For many years, it has been known that mutations in KRAS enhance cellular resistance to ionizing radiation (IR). Clinical data are emerging suggesting that the radioresistance of KRASmut cancer cells that is observed in the laboratory is also seen in patients. However, the underlying mechanisms of mutant KRAS-associated radioresistance is understudied and poorly understood. Cancer stem cells (CSC), also referred to as tumor-initiating cells, may promote metastases development and recurrence after therapy. Generally, cancer cells with CSC-like phenotypes or markers have been found to be radioresistant. Enhanced DNA damage response and repair pathways have been discussed as one underlying mechanism. Interestingly, mutant KRAS has been recently linked to CSC-like phenotypes such as tumor initiation, anchorage independence, and self-renewal. Here, we characterized a radioresistant phenotype associated with KRAS mutated lung cancer cells, xenografts, and patients. CSC-like cells were defined as tumor-initiating cells. Genomic, biochemical, and cell-based assays were used to identify candidates for targeting KRAS mutation-mediated radiation resistance. Radiation resistance is conferred by a CSC-like subpopulation that is characterized by condensed chromatin, high CD133 expression, invasive potential, and tumor-initiating properties. Osteopontin, the gene product of SPP1, promotes aspects of this phenotype including radiation resistance and acts in the same pathway as the EGFR. SPP1/EGFR-dependent chromatin condensation not only protects cells against radiation-induced DNA damage but also downregulates putative negative regulators of CSC-like properties, including CRMP1 and BIM. This phenotype defines a subset of KRAS mutated lung cancers that is enriched for co-occurring TP53 mutations. In conclusions, markers and mechanisms of resistance to radiation therapy in lung and other cancers are poorly characterized. Our data provide novel insight into the aggressive biology of KRAS mutant cancers and have major implications for strategies to overcome the radiation resistance associated with this genotype. Citation Format: Meng Wang, Jing Han, Lynnette Marcar, Josh Black, Qi Liu, Xiangyong Li, Kshithija Nagulapalli, Lecia V. Sequist, Raymond H. Mak, Cyril H. Benes, Theodore S. Hong, Kristin Gurtner, Mechthild Krause, Michael Baumann, Jing X. Kang, Johnathan R. Whetstine, Henning Willers. A SPP1-EGFR pathway links stem-like properties of KRAS-mutated lung cancer to radiation resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4756. doi:10.1158/1538-7445.AM2017-4756

Hyaluronan Production Regulates Metabolic and Cancer Stem-like Properties of Breast Cancer Cells via Hexosamine Biosynthetic Pathway-coupled HIF-1 Signaling

Cancer stem cells (CSCs) represent a small subpopulation of self-renewing oncogenic cells. As in many other stem cells, metabolic reprogramming has been implicated to be a key characteristic of CSCs. However, little is known about how the metabolic features of cancer cells are controlled to orchestrate their CSC-like properties. We recently demonstrated that hyaluronan (HA) overproduction allowed plastic cancer cells to revert to stem cell states. Here, we adopted stable isotope-assisted tracing and mass spectrometry profiling to elucidate the metabolic features of HA-overproducing breast cancer cells. These integrated approaches disclosed an acceleration of metabolic flux in the hexosamine biosynthetic pathway (HBP). A metabolic shift toward glycolysis was also evident by quantitative targeted metabolomics, which was validated by the expression profiles of key glycolytic enzymes. Forced expression of glutamine:fructose-6-phosphate amidotransferase 1 (GFAT1), an HBP rate-limiting enzyme, resembled the results of HA overproduction with regard to HIF-1α accumulation and glycolytic program, whereas GFAT1 inhibition significantly decreased HIF-1α protein level in HA-overproducing cancer cells. Moreover, inhibition of the HBP-HIF-1 axis abrogated HA-driven glycolytic enhancement and reduced the CSC-like subpopulation. Taken together, our results provide compelling evidence that HA production regulates the metabolic and CSC-like properties of breast cancer cells via HBP-coupled HIF-1 signaling.

Iron induces cancer stem cells and aggressive phenotypes in human lung cancer cells.

Evidence has accumulated in support of the critical impact of cancer stem cells (CSCs) behind the chemotherapeutic failure, cancer metastasis, and subsequent disease recurrence and relapse, but knowledge of how CSCs are regulated is still limited. Redox status of the cells has been shown to dramatically influence cell signaling and CSC-like aggressive behaviors. Here, we investigated how subtoxic concentrations of iron, which have been found to specifically induce cellular hydroxyl radical, affected CSC-like subpopulations of human non-small cell lung carcinoma (NSCLC). We reveal for the first time that subchronic iron exposure and higher levels of hydroxyl radical correlated well with increased CSC-like phenotypes. The iron-exposed NSCLC H460 and H292 cells exhibited a remarkable increase in propensities to form CSC spheroids and to proliferate, migrate, and invade in parallel with an increase in level of a well-known CSC marker, ABCG2. We further observed that such phenotypic changes induced by iron were not related to an epithelial-to-mesenchymal transition (EMT). Instead, the sex-determining region Y (SRY)-box 9 protein (SOX9) was substantially linked to iron treatment and hydroxyl radical level. Using gene manipulations, including ectopic SOX9 overexpression and SOX9 short hairpin RNA knockdown, we have verified that SOX9 is responsible for CSC enrichment mediated by iron. These findings indicate a novel role of iron via hydroxyl radical in CSC regulation and its importance in aggressive cancer behaviors and likely metastasis through SOX9 upregulation.

Abstract 3047: Hypoxia leads to deregulation of PI3K/AKT/mTOR signaling in prostate cancer stem cells

Abstract Tumor initiating subpopulations of cancer cells, also known as cancer progenitor or cancer stem cells (CSCs) were recently identified and characterized in prostate cancer. Emerging evidence suggests that hypoxia-inducible factor 1 alpha (HIF1A) contributes to CSC maintenance and influences the PI3K/AKT/mTOR axis, a pathway which is frequently altered in prostate cancer. In our study, we assessed the effects of hypoxia on PI3K/AKT/mTOR signaling in a subset of basal prostate cancer stem cells in a murine model of prostate cancer. First, we successfully isolated Lin-/Sca-1+/CD49f+ basal stem cell subpopulations from primary prostate tumors of TRAMP (transgenic adenocarcinoma of mouse prostate) mice. We observed a consistent decrease in S6 phosphorylation within the CSC subpopulation, in agreement with an mTOR pathway inhibition. However, AKT phosphorylation in the CSC subpopulation was simultaneously elevated, thus prompting further investigations. To analyze the molecular pathways leading to this deregulation, we used a cell line model and isolated Sca-1+/CD49f+ CSC-like subpopulations from the murine prostate cancer cell line TRAMP-C1 and CD44+/CD49f+ CSC-like subpopulations from the androgen independent human prostate cancer cell line DU145. We used sphere formation assays and expression of Notch 1 and Oct 3/4 to confirm the stem and progenitor cell properties of the sorted subpopulations. We could confirm the decrease in mTOR/S6 signaling and identify the upregulation of AKT through IRS-1 mediated feedback loop in prostate CSC-like cells under hypoxic conditions. This AKT/mTOR deregulation was promptly reversed in normoxia, suggestive of the involvement of hypoxia inducible factors. We found elevated HIF1A protein levels as well as increased target gene expression in CSC subpopulations in vitro and in vivo. Further, lentiviral knockdown of HIF1A restored AKT/mTOR activity in CSC-like cells. We could show that CSC-like prostate cancer cells with elevated expression of basal stem cell markers, such as CD44, Sca-1 or CD49f, show a hypoxic deactivation of feedback inhibition on AKT signaling through IRS-1. In light of these results, we propose that deregulation of the PI3K/AKT/mTOR pathway via HIF1A may be critical for cancer stem cell quiescence and maintenance, slowing down cancer stem cell metabolism and growth via mTOR and promoting their survival via AKT signaling. Citation Format: Maximilian Marhold, Erwin Tomasich, Zuzana Pernicová, Radek Fedr, Karel Soucek, Andreas Spittler, Michael Krainer, Peter Horak. Hypoxia leads to deregulation of PI3K/AKT/mTOR signaling in prostate cancer stem cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3047. doi:10.1158/1538-7445.AM2014-3047

Effects of curcumin on stem-like cells in human esophageal squamous carcinoma cell lines

BackgroundMany cancers contain cell subpopulations that display characteristics of stem cells. Because these cancer stem cells (CSCs) appear to provide resistance to chemo-radiation therapy, development of therapeutic agents that target CSCs is essential. Curcumin is a phytochemical agent that is currently used in clinical trials to test its effectiveness against cancer. However, the effect of curcumin on CSCs is not well established. The current study evaluated curcumin-induced cell death in six cancer cell lines derived from human esophageal squamous cell carcinomas. Moreover, these cell lines and the ones established from cells that survived curcumin treatments were characterized.MethodsCell loss was assayed after TE-1, TE-8, KY-5, KY-10, YES-1, and YES-2 cells were exposed to 20–80 μM curcumin for 30 hrs. Cell lines surviving 40 or 60 μM curcumin were established from these six original lines. The stem cell markers aldehyde dehydrogenase-1A1 (ALDH1A1) and CD44 as well as NF-κB were used to compare CSC-like subpopulations within and among the original lines as well as the curcumin-surviving lines. YES-2 was tested for tumorsphere-forming capabilities. Finally, the surviving lines were treated with 40 and 60 μM curcumin to determine whether their sensitivity was different from the original lines.ResultsThe cell loss after curcumin treatment increased in a dose-dependent manner in all cell lines. The percentage of cells remaining after 60 μM curcumin treatment varied from 10.9% to 36.3% across the six lines. The cell lines were heterogeneous with respect to ALDH1A1, NF-κB and CD44 expression. KY-5 and YES-1 were the least sensitive and had the highest number of stem-like cells whereas TE-1 had the lowest. The curcumin-surviving lines showed a significant loss in the high staining ALDH1A1 and CD44 cell populations. Tumorspheres formed from YES-2 but were small and rare in the YES-2 surviving line. The curcumin-surviving lines showed a small but significant decrease in sensitivity to curcumin when compared with the original lines.ConclusionOur results suggest that curcumin not only eliminates cancer cells but also targets CSCs. Therefore, curcumin may be an effective compound for treating esophageal and possibly other cancers in which CSCs can cause tumor recurrence.

Uveal Melanoma Cell Lines Contain Stem-Like Cells That Self-Renew, Produce Differentiated Progeny, and Survive Chemotherapy

Uveal melanoma (UM) cells in high-metastatic risk tumors have an undifferentiated molecular signature indicative of a more primitive cellular phenotype. Given mounting evidence for the existence of cancer stem cells (CSC), the authors investigated whether UM cell lines retain a population of self-renewing tumorigenic cells. Single-cell cloning and spheroid culture studies were used to study the presence of a CSC-like population in two cell lines derived from the primary tumor (Mel270) and metastatic liver lesion (Omm2.5) of the same patient. Mel270 and Omm2.5 cells exhibited distinct clonal morphologies in adherent culture akin to holoclones, meroclones, and paraclones. Holoclones were large colonies of tightly packed small cells, which could be serially passaged (> 10 generations) to produce colonies of all three types; paraclones were small colonies of flattened cells that could be passaged for only one or two generations to produce further paraclones. Mel270 and Omm2.5 cells surviving cisplatin treatment produced significantly more holoclones than untreated cells (P < 0.05), suggesting enrichment for this CSC-like subpopulation. Mel270 and Omm2.5 cells also formed melanomaspheres (MS) when grown at clonal density in nonadherent culture. MS possessed self-renewal capacity to generate further MS and when replated to adherent culture yielded colonies of all three types. Mel270 and Omm2.5 holoclones and MS also demonstrated antigenic heterogeneity expressing markers associated with both a primitive migratory neural crest phenotype, and a more differentiated phenotype. These data suggest the presence in UM cell lines, of a CSC-like subpopulation with enhanced self-renewal and proliferative capabilities that could more appropriately model therapeutic efficacy.

Therapeutic Approaches to Target Cancer Stem Cells

The clinical relevance of cancer stem cells (CSC) remains a major challenge for current cancer therapies, but preliminary findings indicate that specific targeting may be possible. Recent studies have shown that these tumor subpopulations promote tumor angiogenesis through the increased production of VEGF, whereas the VEGF neutralizing antibody bevacizumab specifically inhibits CSC growth. Moreover, nimotuzumab, a monoclonal antibody against the epidermal growth factor receptor (EGFR) with a potent antiangiogenic activity, has been shown by our group to reduce the frequency of CSC-like subpopulations in mouse models of brain tumors when combined with ionizing radiation. These studies and subsequent reports from other groups support the relevance of approaches based on molecular-targeted therapies to selectively attack CSC. This review discusses the relevance of targeting both the EGFR and angiogenic pathways as valid approaches to this aim. We discuss the relevance of identifying better molecular markers to develop drug screening strategies that selectively target CSC.

Abstract 2140: Assessment of curcumin-induced apoptosis and detection of cancer stem cell-like subpopulations in human esophageal cancer cell lines

Abstract Curcumin, the major active ingredient of turmeric, has been shown to kill several types of malignant cells while it has little or no effect on normal cells, suggesting that it may be an effective therapeutic agent. Although some studies have investigated curcumin effects on esophageal cancer cell lines, no studies have characterized the curcumin-surviving cells, an important population that might show resistance to curcumin. There is increasing evidence that many cancers contain subpopulations that display stem cell characteristics. Cancer stem cells (CSCs) appear to play a central role in tumorigenesis, recurrence, metastasis, and resistance to cancer therapy. The aims of the current study were to assess curcumin-induced cell death and detect CSC subpopulations in six human esophageal cancer cell lines (TE-1, TE-8, KY-5, KY-10, YES-1, and YES-2). Aldehyde dehydrogenase-1A1 (ALDH1A1) expression was used as one marker for CSCs. To evaluate curcumin effects, the cell lines were exposed to 20-80 µM of curcumin for 30 hrs. In all six lines, cell death increased continuously with increasing dosage of curcumin based on a standard crystal violet assay. No cells survived at 80 µM, and the range of cell death across the six cell lines was 66 to 89% at 60 µM. To characterize the progeny of cells that survived curcumin, 1×105 cells from each line were exposed to 40-60 µM curcumin for 30 hours, and the colonies that formed from surviving cells were counted. The number of colonies present after 60 µM treatment varied between the cell lines (ranging from 8 to more than one hundred). The Yes-2 cell line did not survive at 60 µM, but produced 15 colonies after 40 µM treatment. Staining with an antibody against ALDH1A1 was used to compare CSC-like subpopulations within and among the original cells lines as well as cell lines established from the curcumin-treated lines. All cell lines were heterogeneous with respect to ALDH1A1 staining, revealing two subpopulations within each line. The intensity of staining was at least two times higher in the population considered to be CSCs. Interestingly, the curcumin-surviving cell lines showed a significant loss in the high-staining population compared to the untreated cells (T-test, p&amp;lt;0.05), suggesting that curcumin not only can kill cancer cells but also CSCs. Thus, esophageal CSCs appear to be highly sensitive to curcumin. Therefore, curcumin may be a more effective compound for esophageal cancer therapy than other chemotherapeutic agents. 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 2140. doi:10.1158/1538-7445.AM2011-2140

Abstract 486: A cancer stem cell population from a head and neck cancer show epithelial to mesenchymal transition properties and resistance to treatment

Abstract Mortality in head and neck squamous cell carcinoma (HNSCC) is high due to the emergence of therapy resistance which led to local and regional recurrences that may originate from resistant cancer stem cells (CSCs). The aim of the present study was to identify cells with CSC characteristics in a HNSCC cell line and evaluate differences in CSC proteins, invasion, migration and in treatment sensitivity to radiation, cisplatin and cetuximab (a antibody blocking epithelial growth factor receptor, EGFR) between subpopulations of cancer cells. A HNSCC cell line established from a larynx cancer at the division of otorhinolaryngology at Linköping University Hospital was used in passages 8-15. Cells were cultured in Keratinocyte Media supplemented with 1% fetal calf serum. Subpopulations of cells were detected after direct immunofluorescense staning of surface CD44 and EGFR. Three different populations (CD44high/EGFRlow, CD44high/EGFRhigh and CD44low/EGFRhigh) were sorted for further investigations using a BD FACSAriaTM II Cytometer. Culturing of sorted cells demonstrated diverse morphology between the populations, where CD44high/EGFRlow cells displayed a spindle shaped morphology compared to CD44low/EGFRhigh. The subpopulations sensitivity to radiation (4Gy), cisplatin (2μg/ml, 1h) or cetuximab (30nM) was analysed 10 days after sorting by a crystal violet assay. Here, CD44high/EGFRlow cells showed the lowest sensitivity to both radiation and cisplatin. A real time PCR array was performed in order to screen for differences in gene expression between the subpopulations. Comparison of the cells with a high contra low CD44 surface expression, showed a change in mRNA levels in 19 genes, 14 up regulated and 5 down regulated. Of these 19 differently expressed genes 13 can be associated to an epithelial to mesenchymal transition (EMT) phenotype (VIM, MMP2, TIMP1, TWIST1, COL3A1, CDH1, CDH2, ITGA5, FN1, FOXC2, WNT5A, WNT5B and SPARC). Two stem cell marker genes (NANOG and SOX1) were slightly up regulated in the CD44high/EGFRlow population compared to CD44low/EGFRhigh. These populations ability for migration and invasion should now be investigated as well as differences in proliferation and clonogenic efficiency. We here identify, in a recently established HNSCC cell line, subpopulations of cells using markers suggested to characterize CSCs. More importantly, the CSC-like subpopulation displayed a lower sensitivity to radiation and cisplatin compared to the control population. 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 486. doi:10.1158/1538-7445.AM2011-486

Abstract 4304: Malignant melanoma cells with aggressive properties are common and not distinguishable by proposed markers for cancer stem cells

Abstract In malignant melanoma, conflicting results have been reported regarding the presence of exclusive cells with enhanced aggressive properties, so called cancer stem cells (CSC), raising a doubt whether melanoma follows a CSC model and is hierarchically organized. By characterizing melanoma cells from short-term cell cultures, xenografts and patient biopsies, we aim to collect further evidence either to: i) support the existence of distinct CSC-like subpopulations, or ii) strengthen a notion about the common presence of cells demonstrating aggressive behavior and lack of cellular hierarchy in melanoma. By using individual-cell assays, we have shown that a large fraction (up to 60 %) of random single melanoma cells display high clonogenicity and self-renewal i.e. properties associated with tumorigenic potential. In search for a marker for such clonogenic/tumorigenic cells, we found that melanomas often harbor a large distinct subpopulation with elevated activity of Aldehyde Dehydrogenase (ALDH), a proposed marker for CSC-like cells in some hierarchically organized cancers. Interestingly, ALDH activity in melanoma patient biopsies seemed to correlate to the expression of melanoma-associated-antigen, HMW-MAA, which is linked to tumor progression. Furthermore, in vivo ALDH+ melanoma cells could convert to ALDH−, while the opposite conversion was rare, indicating a certain “cellular hierarchy” with respect to the ALDH phenotype. However, comparison of ALDH+ and ALDH− cells revealed that both subpopulations are highly clonogenic, tumorigenic and resistant to drugs proposed for melanoma therapy, DTIC and HGS-ETR2. This suggests that in malignant melanoma, ALDH, likewise earlier investigated “surface markers” of CSCs, does not distinguish cells with enhanced biological aggressiveness. In conclusion, melanoma cells exhibiting properties linked to aggressive phenotype are common and not restricted to subpopulations expressing proposed “CSC markers”, which contradicts the traditional view of CSCs. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 4304.