Cancer Stem Cell Gene Signature Research Articles

KITlow Cells Mediate Imatinib Resistance in Gastrointestinal Stromal Tumor.

Gastrointestinal stromal tumor (GIST) is commonly driven by oncogenic KIT mutations that are effectively targeted by imatinib (IM), a tyrosine kinase inhibitor (TKI). However, IM does not cure GIST, and adjuvant therapy only delays recurrence in high-risk tumors. We hypothesized that GIST contains cells with primary IM resistance that may represent a reservoir for disease persistence. Here, we report a subpopulation of CD34+KITlow human GIST cells that have intrinsic IM resistance. These cells possess cancer stem cell-like expression profiles and behavior, including self-renewal and differentiation into CD34+KIThigh progeny that are sensitive to IM treatment. We also found that TKI treatment of GIST cell lines led to induction of stem cell-associated transcription factors (OCT4 and NANOG) and concomitant enrichment of the CD34+KITlow cell population. Using a data-driven approach, we constructed a transcriptomic-oncogenic map (Onco-GPS) based on the gene expression of 134 GIST samples to define pathway activation during GIST tumorigenesis. Tumors with low KIT expression had overexpression of cancer stem cell gene signatures consistent with our in vitro findings. Additionally, these tumors had activation of the Gas6/AXL pathway and NF-κB signaling gene signatures. We evaluated these targets in vitro and found that primary IM-resistant GIST cells were effectively targeted with either single-agent bemcentinib (AXL inhibitor) or bardoxolone (NF-κB inhibitor), as well as with either agent in combination with IM. Collectively, these findings suggest that CD34+KITlow cells represent a distinct, but targetable, subpopulation in human GIST that may represent a novel mechanism of primary TKI resistance, as well as a target for overcoming disease persistence following TKI therapy.

Expression of cancer stem cell markers in tall cell variant papillary thyroid cancer identifies a molecular profile predictive of recurrence in classic papillary thyroid cancer

BackgroundTall cell variant of papillary thyroid carcinoma is an aggressive subtype of papillary thyroid carcinoma. We examined expression of cancer stem cell markers in tall cell variant compared with other well-differentiated thyroid cancers. MethodsExpression of cancer stem cell markers was examined in 572 thyroid tumors from The Cancer Genome Atlas Thyroid Cancer database and tall cell variant and papillary thyroid carcinoma tumors by immunohistochemistry. ResultsExpression of the PROM1 gene, encoding the cancer stem cell marker CD133, was elevated in tall cell variant compared to classic papillary thyroid carcinoma in a large cohort of unmatched samples from The Cancer Genome Atlas Thyroid Cancer database (P < .001). By immunohistochemistry in age and stage matched samples, CD133 protein was confirmed to be significantly increased in tall cell variant versus classic papillary thyroid carcinoma (P = .006). Analyzing all thyroid cancers, high PROM1 expression was associated with worse disease-specific survival. Optimal cutoffs were determined to define a tall cell variant-like cancer stem cell signature characterized by high PROM1, high ALDH1A3, and low CD24 expression. Classic papillary thyroid carcinoma with a tall cell variant-like gene signature had worse recurrence disease-free survival compared to classic papillary thyroid carcinoma with a non-tall cell variant signature (P = .02). ConclusionTall cell variant of papillary thyroid carcinoma has increased expression of cancer stem cell markers compared to classic papillary thyroid carcinoma. The tall cell variant-like cancer stem cell gene signature identified a molecular subtype of classic papillary thyroid carcinoma that has a worse recurrence-free survival.

CD44 splice isoform switching determines breast cancer stem cell state.

Although changes in alternative splicing have been observed in cancer, their functional contributions still remain largely unclear. Here we report that splice isoforms of the cancer stem cell (CSC) marker CD44 exhibit strikingly opposite functions in breast cancer. Bioinformatic annotation in patient breast cancer in The Cancer Genome Atlas (TCGA) database reveals that the CD44 standard splice isoform (CD44s) positively associates with the CSC gene signatures, whereas the CD44 variant splice isoforms (CD44v) exhibit an inverse association. We show that CD44s is the predominant isoform expressed in breast CSCs. Elimination of the CD44s isoform impairs CSC traits. Conversely, manipulating the splicing regulator ESRP1 to shift alternative splicing from CD44v to CD44s leads to an induction of CSC properties. We further demonstrate that CD44s activates the PDGFRβ/Stat3 cascade to promote CSC traits. These results reveal CD44 isoform specificity in CSC and non-CSC states and suggest that alternative splicing provides functional gene versatility that is essential for distinct cancer cell states and thus cancer phenotypes.

High-Throughput Microfluidic Labyrinth for the Label-free Isolation of Circulating Tumor Cells.

We present "Labyrinth," a label-free microfluidic device to isolate circulating tumor cells (CTCs) using the combination of long loops and sharp corners to focus both CTCs and white blood cells (WBCs) at a high throughput of 2.5mL/min. The high yield (>90%) and purity (600 WBCs/mL) of Labyrinth enabled us to profile gene expression in CTCs. As proof of principle, we used previously established cancer stem cell gene signatures to profile single cells isolated from the blood of breast cancer patients. We observed heterogeneous subpopulations of CTCs expressing genes for stem cells, epithelial cells, mesenchymal cells, and cells transitioning between epithelial and mesenchymal. Labyrinth offers a cell-surface marker-independent single-cellisolation platform to study heterogeneous CTC subpopulations.

Abstract 2898: ST8SIA1 is up-regulated in triple negative breast cancer and its expression is positively correlated with TP53 mutations and a cancer stem cell gene signature

Abstract We discovered ganglioside GD2 as a breast cancer stem cell (BCSCs) marker in triple negative breast cancer (TNBC). GD2 biosynthesis is tightly regulated by the enzyme ST8SIA1 (GD3 synthase). However, expression of ST8SIA and its association with different breast cancer sub-types is not known. Here we hypothesize that ST8SIA1 is up-regulated in TNBC or “basal-type” breast tumors and associated with stemness in primary breast tumors. To investigate ST8SIA1 expression in primary tumors, we analyzed RNAseq data from the cancer genome atlas (TCGA) data base, which includes data from 1105 primary and metastatic breast tumors as well as adjacent normal tissues. We found that ST8SIA1 expression varied widely among different breast tumors. Interestingly, “basal-type” tumors expressed the highest levels of ST8SIA1 compared to all other types of breast cancers including luminal-A or luminal-B or HER2-enriched tumors (p &amp;lt; 0.01). In addition, TNBC (n = 115) expressed 4.6-fold higher ST8SIA1 levels compared to hormone receptors positive tumors including ER+ or PR+ or HER2+ tumors (n = 852, p&amp;lt;0.001). Survival analysis by log-rank test indicated that patients with ST8SIA1high tumors survive shorter (median survival 2.6 years) compared to patients with ST8SIA1low tumors (median survival 4.3 years). Next, we investigates the association between ST8SIA1expression and the most commonly mutated genes in breast cancer. We chose the top 20 most frequently mutated genes in TCGA dataset and examined their correlation with ST8SIA1 mRNA expression. We found that, among the top 20 mutations, TP53 had a very strong positive correlation with ST8SIA1 expression (p &amp;lt; 0.00001). In fact, the expression of ST8SIA1 was &amp;gt; 2-fold higher in TP53-mutated compared to wild type tumors. The other positively correlated mutation was a nuclear envelop protein called the spectrin repeat containing protein (nuclear envelope 1 or SYNE1; p &amp;lt; 0.05). Mutations in GATA3 were the most negatively correlated (p &amp;lt; 0.001) with ST8SIA1 expression. Interestingly, GATA3 plays a role in epithelial cell differentiation in the mammary gland, supporting the notion that ST8SIA1 is a stem cell-associated gene. In addition, correlation of ST8SIA1 mRNA expression with other genes revealed that FOXA1, the protein which is co-expressed with GATA3 and serves as negative predictor of “basal-type” of breast cancer, was down regulated in ST8SIA1high tumors. In conclusion, ST8SIA1 is associated with “basal-type” TNBC tumors and it has a strong positive correlation with TP53 mutations and negative correlation with GATA3 mutations. Knowing that TP53 mutations have a major role in tumorigenesis and drug resistance, these data suggest that ST8SIA1 could be a potential therapeutic target in patients with TP53-mutant TNBC. Citation Format: V. Lokesh Battula, Yuanqing Yan, Khoa Nguyen, Kim-Anh Do, Michael Andreeff. ST8SIA1 is up-regulated in triple negative breast cancer and its expression is positively correlated with TP53 mutations and a cancer stem cell gene signature [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 2898. doi:10.1158/1538-7445.AM2017-2898

Abstract 1849: The role of leptin signaling in the promotion of obesity-associated tumorigenesis and cancer stem cell characteristics in a transgenic mouse model of basal-like breast cancer

Abstract Background: Epidemiological studies have linked obesity to a greater risk of breast cancer-specific mortality. This association may be mediated in part by enhanced cancer stem cell (CSC) enrichment, as CSCs are thought to be the primary drivers of tumor initiation, growth, and metastasis. We and others have have demonstrated that obesity promotes CSC enrichment in pre-clinical models of basal-like breast cancer, but only when leptin signaling is present. These findings, coupled with evidence that elevated serum leptin and breast tumor leptin receptor expression are linked to a worse breast cancer prognosis, suggest that leptin-induced CSC enrichment may be a key contributor to obesity-associated breast cancer progression. Methods: MMTV-Wnt-1 transgenic mice, which develop spontaneous basal-like mammary tumors, were fed a control diet (10% kcal from fat) or a diet-induced obesity regimen (DIO, 60% kcal from fat). Mice were euthanized when tumors reached 1.5 cm in diameter. RNA sequencing was performed on 4 tumors/diet group. Serum hormones and cytokines were measured via luminex-based assay. ALDH activity, a putative CSC marker, was quantified by an ALDH Activity Assay Kit (abcam). Quantitative RT-PCR was used to determine expression of CSC-related genes, leptin, and the leptin receptor. Two mammary tumor cell lines isolated from the MMTV-Wnt-1 model (M-Wnt and E-Wnt cells) and human MDA-MB-231 were exposed in vitro to pooled serum from DIO or control mice (2% concentration), and cell viability, migration, invasion and expression of CSC related genes were measured. Results: Tumor incidence and growth rate were greater in DIO versus control mice. RNA sequencing revealed an upregulation in a CSC gene signature in tumors from DIO versus control mice. Significant upregulation in several of these genes, including aldh1a, oct4, twist1, twist2, and akt3, was confirmed by qRT-PCR (P &amp;lt; 0.05). ALDH activity in the DIO (relative to control) tumors was also significantly increased (P &amp;lt; 0.05). Serum levels and mammary tissue expression of leptin and tumor expression of the leptin receptor were significantly elevated in DIO versus control mice (P &amp;lt; 0.05). Exposure to serum from DIO mice significantly increased (relative to control) M-Wnt, E-Wnt, and MDA-MB-231 cell viability, migration, invasion, and expression of CSC-related genes in vitro (P &amp;lt; 0.05). The role of leptin signaling in these effects and in vitro measures of CSC enrichment will be explored through additional in experiments. Conclusions: Obesity promotes MMTV-Wnt-1 mammary tumor incidence and growth rate, possibly via CSC enrichment. Preliminary findings suggest increased leptin signaling may underlie obesity-associated CSC enrichment and may contribute to the breast cancer burden in obese women. Citation Format: Subreen A. Khatib, Laura W. Bowers, Emily L. Rossi, Shannon B. McDonell, Claire G. Lineberger, David A. Cavazos, Linda A. deGraffenried, Stephen D. Hursting. The role of leptin signaling in the promotion of obesity-associated tumorigenesis and cancer stem cell characteristics in a transgenic mouse model of basal-like breast cancer [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 1849. doi:10.1158/1538-7445.AM2017-1849

Abstract S4-03: Identification of a notch-driven breast cancer stem cell gene signature for anti-notch therapy in an ER+ presurgical window model

Abstract Background: Resistance to endocrine therapy (ET; tamoxifen or aromatase inhibitors, AI) for ER+ breast cancer is a major cause of mortality and new treatment paradigms are needed. Cancer stem cells drive breast cancer growth and are resistant to standard therapy. Notch signaling aids survival of these resistant stem cells and is inhibited by gamma-secretase inhibitors (GSI). We showed combining GSI with ET in mice caused shrinkage of breast cancer tumors. A presurgical window biomarker modulation model was used to confirm this discovery in humans. Methods: The GSI MK-0752 was added to ET in patients before definitive surgery (ClinTrials.gov NCT00756717). There were 3 biopsies: day 0 (prestudy), day 14 (after ET alone), and day 25 at definitive surgery (after continued ET plus MK-0752, 350 mg orally 3d on, 4d off, 3d on). Biopsies were analyzed for genes increased or decreased by GSI, to confirm that Notch and cancer stem cell pathways were inhibited. Real-time PCR was used to validate expression of genes identified in pathway analyses of microarray datasets generated from the biopsies. Mammosphere-forming assays were performed to confirm that ET+GSI impacts breast cancer stem cells. The qRT-PCR data were evaluated using ANOVA with repeated measures and ANOVA was performed on mammosphere results. Results: The accrual goal was met and therapy well-tolerated in 20 evaluable women (PSABCS 2011, abs# S1-5). Of 33 genes identified by analysis of expression microarrays, 19 genes (FDR&amp;lt;8%) were impacted significantly by GSI+ ET (3 increased, 16 decreased) compared to initial biopsy and/or ET alone. Genes with increased expression were DAXX, NOXA (both pro-apoptotic) and LNFG (tumor suppressor). Six of 16 genes that decreased (NOTCH1, NOTCH4, HEYL, HES1, HES5, and HEY2) are Notch pathway-associated genes. The GSI decreased expression of 3 genes from cell cycle and proliferative pathways (Ki67, CCND1, CCNA2) and inhibited 2 genes expressed in cancer stem cells (RUNX1 and ALDH1A1). Five genes directly/indirectly regulated by Notch were decreased by GSI (RICTOR, RPTOR, MMP7, ADAM19, and PRH). Estrogen deprivation for 3 days, mimicking short exposure to an AI, increased mammosphere-forming ability of ER+ breast cancer cells more than 2 fold. The GSI MRK-003 blocked this mammosphere formation by 95%-98%. Conclusions: A 7-day course of the GSI MK-0752 added to ET in the presurgical window had significant biomarker responses: decrease in Notch signaling, cancer stem cell genes, proliferation-associated genes, the mTORC1 and 2 complex genes RICTOR and RPTOR, metalloproteinases that promote metastasis, and PRH; as well as increase in 3 key genes that promote apoptosis and tumor suppression. These results suggest that 1) GSI inhibited the intended Notch pathway, 2) putative breast cancer stems cells can be targeted by this strategy, and 3) the biomarkers identified create a gene signature for anti-Notch therapy in ER+ breast cancer. Validation of efficacy of the GSI+ET therapy combination and this gene signature in a clinical trial is planned. Support: Breast Cancer Research Foundation (research grant), Merck Oncology (drug/arrays), Swim Across America (clinical trial costs), and DOD BC073237 (KRC). Citation Format: Kathy S Albain, Andrei Y Zlobin, Kyle R Covington, Brian T Gallahger, Susan G Hilsenbeck, Cheryl M Czerlanis, Shelly Lo, Patricia A Robinson, Ellen R Gaynor, Constantine Godellas, Davide Bova, Kathy Czaplicki, Barbara Busby, Patrick J Stiff, Suzanne AW Fuqua, Lucio Miele, Clodia Osipo. Identification of a notch-driven breast cancer stem cell gene signature for anti-notch therapy in an ER+ presurgical window model [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr S4-03.

Spontaneous development of hepatocellular carcinoma with cancer stem cell properties in PR-SET7-deficient livers.

PR-SET7-mediated histone 4 lysine 20 methylation has been implicated in mitotic condensation, DNA damage response and replication licensing. Here, we show that PR-SET7 function in the liver is pivotal for maintaining genome integrity. Hepatocyte-specific deletion of PR-SET7 in mouse embryos resulted in G2 phase arrest followed by massive cell death and defect in liver organogenesis. Inactivation at postnatal stages caused cell duplication-dependent hepatocyte necrosis, accompanied by inflammation, fibrosis and compensatory growth induction of neighboring hepatocytes and resident ductal progenitor cells. Prolonged necrotic regenerative cycles coupled with oncogenic STAT3 activation led to the spontaneous development of hepatic tumors composed of cells with cancer stem cell characteristics. These include a capacity to self-renew in culture or in xenografts and the ability to differentiate to phenotypically distinct hepatic cells. Hepatocellular carcinoma in PR-SET7-deficient mice displays a cancer stem cell gene signature specified by the co-expression of ductal progenitor markers and oncofetal genes.

ARID3B increases ovarian tumor burden and is associated with a cancer stem cell gene signature.

Ovarian cancer is the most deadly gynecological malignancy since most patients have metastatic disease at the time of diagnosis. Therefore, identification of critical pathways that contribute to ovarian cancer progression is necessary to yield novel therapeutic targets. Recently we reported that the DNA binding protein ARID3B is overexpressed in human ovarian tumors. To determine if ARID3B has oncogenic functions in vivo, ovarian cancer cell lines stably expressing ARID3B were injected intraperitoneally into nude mice. Overexpression of ARID3B increased tumor burden and decreased survival. To assess how ARID3B contributes to the increased tumor growth in vivo, we identified ARID3B induced genes in tumor ascites cells. ARID3B induced expression of genes associated with metastasis and cancer stem cells (CD44, LGR5, PROM1 (CD133), and Notch2). Moreover, ARID3B increased the number of CD133+ (a cancer stem cell marker) cells compared to control cells. The increase in CD133+ cells resulting from ARID3B expression was accompanied by enhanced paclitaxel resistance. Our data demonstrate that ARID3B boosts production CD133+ cells and increases ovarian cancer progression in vivo.

Effective depletion of hepatic cancer stem-like cells by NF-kB mediated inhibiton of histone deacetylases

Over the past few years, compelling evidence has emerged in support of the hierarchic cancer stem cell (CSC) model for many solid tumors, including hepatocellular cancers. We have recently identified a CSC gene signature that exhibited a pernicious interaction with a variety of key oncogenic and common stemness pathways. Among these common pathways, activation of NF-kB signaling was consistently observed in human liver CSCs. Based on these data, we hypothesized that NF-kB may be a specific therapeutic target against CSC. In this study, we evaluated whether the inhibition of NF-kB would selectively deplete liver CSC.

Introduction of SV40ER and hTERT into mammospheres generates breast cancer cells with stem cell properties

Emerging evidence suggests that cancers arise in stem/progenitor cells. Yet, the requirements for transformation of these primitive cells remains poorly understood. In this study, we have exploited the 'mammosphere' system that selects for primitive mammary stem/progenitor cells to explore their potential and requirements for transformation. Introduction of Simian Virus 40 Early Region and hTERT into mammosphere-derived cells led to the generation of NBLE, an immortalized mammary epithelial cell line. The NBLEs largely comprised of bi-potent progenitors with long-term self-renewal and multi-lineage differentiation potential. Clonal and karyotype analyses revealed the existence of heterogeneous population within NBLEs with varied proliferation, differentiation and sphere-forming potential. Significantly, injection of NBLEs into immunocompromised mice resulted in the generation of invasive ductal adenocarcinomas. Further, these cells harbored a sub-population of CD44(+)/CD24(-) fraction that alone had sphere- and tumor-initiating potential and resembled the breast cancer stem cell gene signature. Interestingly, prolonged in vitro culturing led to their further enrichment. The NBLE cells also showed increased expression of stemness and epithelial to mesenchymal transition markers, deregulated self-renewal pathways, activated DNA-damage response and cancer-associated chromosomal aberrations-all of which are likely to have contributed to their tumorigenic transformation. Thus, unlike previous in vitro transformation studies that used adherent, more differentiated human mammary epithelial cells our study demonstrates that the mammosphere-derived, less-differentiated cells undergo tumorigenic conversion with only two genetic elements, without requiring oncogenic Ras. Moreover, the striking phenotypic and molecular resemblance of the NBLE-generated tumors with naturally arising breast adenocarcinomas supports the notion of a primitive breast cell as the origin for this subtype of breast cancer. Finally, the NBLEs represent a heterogeneous population of cells with striking plasticity, capable of differentiation, self-renewal and tumorigenicity, thus offering a unique model system to study the molecular mechanisms involved with these processes.

Human hepatic cancer stem cells are characterized by common stemness traits and diverse oncogenic pathways

Epigenetic mechanisms play critical roles in stem cell biology by maintaining pluripotency of stem cells and promoting differentiation of more mature derivatives. If similar mechanisms are relevant for the cancer stem cell (CSC) model, then epigenetic modulation might enrich the CSC population, thereby facilitating CSC isolation and rigorous evaluation. To test this hypothesis, primary human cancer cells and liver cancer cell lines were treated with zebularine (ZEB), a potent DNA methyltransferase-1 inhibitor, and putative CSCs were isolated using the side population (SP) approach. The CSC properties of ZEB-treated and untreated subpopulations were tested using standard in vitro and in vivo assays. Whole transcriptome profiling of isolated CSCs was performed to generate CSC signatures. Clinical relevance of the CSC signatures was evaluated in diverse primary human cancers. Epigenetic modulation increased frequency of cells with CSC properties in the SP fraction isolated from human cancer cells as judged by self-renewal, superior tumor-initiating capacity in serial transplantations, and direct cell tracking experiments. Integrative transcriptome analysis revealed common traits enriched for stemness-associated genes, although each individual CSC gene expression signature exhibited activation of different oncogenic pathways (e.g., EGFR, SRC, and MYC). The common CSC signature was associated with malignant progression, which is enriched in poorly differentiated tumors, and was highly predictive of prognosis in liver and other cancers. Epigenetic modulation may provide a tool for prospective isolation and in-depth analysis of CSC. The liver CSC gene signatures are defined by a pernicious interaction of unique oncogene-specific and common stemness traits. These data should facilitate the identifications of therapeutic tools targeting both unique and common features of CSCs.

Abstract A29: Discovery and preclinical to clinical evaluation of biomarkers for Anti-DLL4, a monoclonal antibody targeting cancer stem cells

Abstract Blockade of DLL4 signaling reduces tumor growth in preclinical human xenograft models by disrupting productive angiogenesis and reducing proliferation of tumor cells. Importantly in these models, blocking DLL4 also reduces cancer stem cell (CSC) frequency in multiple solid tumor types. Currently, there exists an important need to identify and evaluate pharmacodynamic biomarkers that indicate the biological activity of agents, such as anti-DLL4, that are in clinical studies. Towards this aim, we used numerous human xenograft models derived from primary human tumors to identify and characterize biomarkers for anti-DLL4. Here, we present data from these studies and describe anti-DLL4 biomarkers in tumors, in hair follicles, and in blood. We show that several vascular-related genes are significantly upregulated by anti-DLL4 in tumors including, Egfl7, Apln, Cldn5, and Notch4. We also demonstrate significant downregulation of CSC gene signatures and modulation of differentiation genesets. A subset of the identified markers were validated at the protein level and also tested in a human skin graft model, where the human tumor is engrafted in a human microenvironment/vasculature. Finally, a DLL4-Notch gene signature was identified that showed a significant association with breast cancer survival using publicly available cancer gene sets. In surrogate tissues, we identified anti-DLL4 biomarkers in blood and in hair follicles. These markers from surrogate tissues were translated into a phase I trial of Anti-DLL4 antibody (OMP-21M18), and an interim analysis of this data demonstrates that OMP-21M18 affects the Notch pathway in patient samples. Citation Information: Clin Cancer Res 2010;16(14 Suppl):A29.

Patents Related to Cancer Stem Cell Research

Cancer has long been viewed as a heterogeneous population of cells. Cancerous cells may often originate from the transformation of normal stem cells, similar signaling pathways may regulate self-renewal in stem cells and cancer cells. The concept that cancer might arise from a rare population of cells with stem cell properties was proposed for more than a century. Cancer stem cell hypothesis has began to be accepted recently due to the advances in stem cell biology and the development of new animal models to measure self-renewal that drive tumorigenesis. Cancer stem cells have been identified and purified in a variety of tumors (blood, breast, brain, colon, lung, pancreas) using unique stem cell markers such as CD44, CD133 and aldehyde dehydrogenases. Cancer stem cell gene signatures have been examined. This review will discuss the evolution of cancer stem cell research and summarize the recent patents related to the cancer stem cell markers, the methods to detect and modulate cancer stem cells and cancer stem cell-targeted treatment. With the advances in cancer stem cell research, the new patent applications, particularly the new drugs on cancer stem cells treatments are expected to be increasing.

Cancer stem cell gene signature of ovarian tumor side populations

5546 Background: Recent studies support the concept that cancer as a disease is driven by a small subset of cells, cancer stem cells. These cells can initiate tumor growth and re-initiate tumor expansion after therapy. The aim of this study is to generate a cancer stem cell gene expression signature from isolated Side Populations (SP) of fresh ascites obtained from women with high-grade advanced stage papillary serous ovarian adenocarcinoma. Methods: All patient specimens were collected/studied under protocols approved by the institutional review board of the parent institution. Gene expression profiling using Affymetrix U133 2.0 Plus microarray was performed on SP and Main Populations (MP) isolated by Hoechst staining/FACS method. Paired T-test analyses (p<0.01) identified differentially expressed genes between SP and MP. The microarray data was validated using qRT-PCR. Results: 446 differentially expressed genes (138 up- and 302 down-regulated) were found between all 10 SP/MP pairs. Microarray validation on 19 randomly selected differentially regulated genes, showed 17/19 (89.5%) genes has robust correlations between microarray and qRT-PCR expression data. The gene signature was enriched for genes in GO biological processes of cell cycle, transport, apoptosis, regulation of translation/transcription, signal transduction, and cell proliferation. Further data mining for biologically relevant processes using Pathway Studio 5.0 identified overexpressed genes in SP related to functional cancer stem cell-like phenotypes of cell survival (PAWR), proliferation (EPHB), and apoptosis (AKT). WNT signaling pathway genes (FZD) were downregulated in SP, and genes implicated in normal stem cell function (NUP, ST3GAL, LTBP) were upregulated in SP. These results highlight the ‘stemness function‘ of the ovarian cancer SP gene signature. Conclusions: We generated an expression profile from SP enriched for cancer stem cells from ascites from ovarian cancer patients. The ‘stemness‘ nature of the SP gene signature was revealed by the identification of several stem cell-related genes and genes involved in the WNT -signaling pathway. These genes may be key players involved in the mechanisms controlling ovarian cancer stem cell functions, and may represent potential therapeutic targets. No significant financial relationships to disclose.