Karen M. Hite

Complex display of putative tumor stem cell markers in the NCI60 tumor cell line panel.

Tumor stem cells or cancer initiating cells (CICs) are single tumor cells that can regenerate a tumor or a metastasis. The identification and isolation of CICs remain challenging, and a variety of putative CIC markers have been described. We hypothesized that cell lines of the NCI60 panel contain CICs and express putative CIC markers. We investigated expression of putative CIC surface markers (CD15, CD24, CD44, CD133, CD166, CD326, PgP) and the activity of aldehyde dehydrogenase in the NCI60 panel singly and in combination by six‐color fluorescence‐activated cell sorting analysis. All investigated markers were expressed in cell lines of the NCI60 panel. Expression levels of individual markers varied widely across the 60 cell lines, and neither single marker expression nor simple combinations nor co‐expression patterns correlated with the colony‐formation capacity of cell lines. Rather, marker expression patterns correlated with tumor types in multidimensional analysis. Whereas some expression patterns correlated with tumor entities such as basal breast cancer, other expression patterns occurred across different tumor types and largely related to expression of a more mesenchymal phenotype in individual breast, lung, renal, and melanoma cell lines. Our data for the first time demonstrate that tumor cell lines display CIC markers in a complex pattern that relates to the tumor type. The complexity and tumor type specificity of marker display creates challenges for the application of cell sorting and other approaches to isolation of putative tumor stem cell populations and suggests that therapeutic targeting strategies will need to take this into account. STEM CELLS 2010;28:649–66028:649–660

Identification of CBX3 and ABCA5 as Putative Biomarkers for Tumor Stem Cells in Osteosarcoma

Recently, there has been renewed interest in the role of tumor stem cells (TSCs) in tumorigenesis, chemoresistance, and relapse of malignant tumors including osteosarcoma. The potential exists to improve osteosarcoma treatment through characterization of TSCs and identification of therapeutic targets. Using transcriptome, proteome, immunophenotyping for cell-surface markers, and bioinformatic analyses, heterogeneous expression of previously reported TSC or osteosarcoma markers, such as CD133, nestin, POU5F1 (OCT3/4), NANOG, SOX2, and aldehyde dehydrogenase, among others, was observed in vitro. However, consistently significantly lower CD326, CD24, CD44, and higher ABCG2 expression in TSC-enriched as compared with un-enriched osteosarcoma cultures was observed. In addition, consistently higher CBX3 expression in TSC-enriched osteosarcoma cultures was identified. ABCA5 was identified as a putative biomarker of TSCs and/or osteosarcoma. Lastly, in a high-throughput screen we identified epigenetic (5-azacytidine), anti-microtubule (vincristine), and anti-telomerase (3,11-difluoro-6,8,13-trimethyl- 8H-quino [4,3,2-kl] acridinium methosulfate; RHPS4)-targeted therapeutic agents as candidates for TSC ablation in osteosarcoma.

Cell type-specific, topoisomerase II-dependent inhibition of hypoxia-inducible factor-1alpha protein accumulation by NSC 644221.

Purpose: The discovery and development of small-molecule inhibitors of hypoxia-inducible factor-1 (HIF-1) is an attractive, yet challenging, strategy for the development of new cancer therapeutic agents. Here, we report on a novel tricyclic carboxamide inhibitor of HIF-1α, NSC 644221. Experimental Design: We investigated the mechanism by which the novel compound NSC 644221 inhibited HIF-1α. Results: NSC 644221 inhibited HIF-1–dependent, but not constitutive, luciferase expression in U251-HRE and U251-pGL3 cells, respectively, as well as hypoxic induction of vascular endothelial growth factor mRNA expression in U251 cells. HIF-1α, but not HIF-1β, protein expression was inhibited by NSC 644221 in a time- and dose-dependent fashion. Interestingly, NSC 644221 was unable to inhibit HIF-1α protein accumulation in the presence of the proteasome inhibitors MG132 or PS341, yet it did not directly affect the degradation of HIF-1α as shown by experiments done in the presence of cyclohexamide or pulse-chase labeling using [35S]methionine. In contrast, NSC 644221 decreased the rate of HIF-1α translation relative to untreated controls. Silencing of topoisomerase (topo) IIα, but not topo I, by specific small interfering RNA completely blocked the ability of NSC 644221 to inhibit HIF-1α. The data presented show that topo II is required for the inhibition of HIF-1α by NSC 644221. Furthermore, although NSC 644221 induced p21 expression, γH2A.X, and G2-M arrest in the majority of cell lines tested, it only inhibited HIF-1α in a distinct subset of cells, raising the possibility of pathway-specific “resistance” to HIF-1 inhibition in cancer cells. Conclusions: NSC 644221 is a novel HIF-1 inhibitor with potential for use as both an analytic tool and a therapeutic agent. Our data provide a strong rationale for pursuing the preclinical development of NSC 644221 as a HIF-1 inhibitor.

Cysteine 81 is critical for the interaction of S100A4 and myosin-IIA.

Overexpression of S100A4, a member of the S100 family of Ca(2+)-binding proteins, is associated with a number of human pathologies, including fibrosis, inflammatory disorders, and metastatic disease. The identification of small molecules that disrupt S100A4/target interactions provides a mechanism for inhibiting S100A4-mediated cellular activities and their associated pathologies. Using an anisotropy assay that monitors the Ca(2+)-dependent binding of myosin-IIA to S100A4, NSC 95397 was identified as an inhibitor that disrupts the S100A4/myosin-IIA interaction and inhibits S100A4-mediated depolymerization of myosin-IIA filaments. Mass spectrometry demonstrated that NSC 95397 forms covalent adducts with Cys81 and Cys86, which are located in the canonical target binding cleft. Mutagenesis studies showed that covalent modification of just Cys81 is sufficient to inhibit S100A4 function with respect to myosin-IIA binding and depolymerization. Remarkably, substitution of Cys81 with serine or alanine significantly impaired the ability of S100A4 to promote myosin-IIA filament disassembly. As reversible covalent cysteine modifications have been observed for several S100 proteins, we propose that modification of Cys81 may provide an additional regulatory mechanism for mediating the binding of S100A4 to myosin-IIA.

A Small Molecule (Pluripotin) as a Tool for Studying Cancer Stem Cell Biology: Proof of Concept

Background Cancer stem cells (CSC) are thought to be responsible for tumor maintenance and heterogeneity. Bona fide CSC purified from tumor biopsies are limited in supply and this hampers study of CSC biology. Furthermore, purified stem-like CSC subpopulations from existing tumor lines are unstable in culture. Finding a means to overcome these technical challenges would be a useful goal. In a first effort towards this, we examined whether a chemical probe that promotes survival of murine embryonic stem cells without added exogenous factors can alter functional characteristics in extant tumor lines in a fashion consistent with a CSC phenotype. Methodology/Principal Findings The seven tumor lines of the NCI60 colon subpanel were exposed to SC-1 (pluripotin), a dual kinase and GTPase inhibitor that promotes self-renewal, and then examined for tumorigenicity under limiting dilution conditions and clonogenic activity in soft agar. A statistically significant increase in tumor formation following SC-1 treatment was observed (p<0.04). Cloning efficiencies and expression of putative CSC surface antigens (CD133 and CD44) were also increased. SC-1 treatment led to sphere formation in some colon tumor lines. Finally, SC-1 inhibited in vitro kinase activity of RSK2, and another RSK2 inhibitor increased colony formation implicating a role for this kinase in eliciting a CSC phenotype. Conclusions/Significance These findings validate a proof of concept study exposure of extant tumor lines to a small molecule may provide a tractable in vitro model for understanding CSC biology.

Abstract 5202: Human colon cancer cell lines contain subsets of cells with the capacity to initiate highly prolific clonal growth in soft agar culture and to form transplantable tumor xenografts in vivo

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Various studies underscore the key role of cancer ‘stem cells’ in the initiation of experimental tumor xenografts from cells derived from patient tumor specimens. Implantation of limited numbers of cancer cells – even ‘single cells’ – which exhibit putative cancer ‘stem cell’ markers were reported to form tumors in mice, although several months were required to achieve palpable tumor masses. In the current study we assessed whether established human colon cancer cell lines still possess the functional capacity to initiate tumor formation from ‘single cell’ preparations. Results indicate that each of seven colon cancer cell lines was capable of soft agar colony formation in RPMI-1640 medium containing either 5-10% FBS or 15% serum substitute. Inoculation of 96-well plates by single cell sorting resulted in highly prolific colony formation by a subset of cells derived from each cell line. While the large majority of colonies achieving a size >60μ in diameter exhibited growth arrest within 3 weeks (<<10 cell mass doublings), a minority of clones (<2%) exhibited sustained growth over the course of 3 – 8 weeks. Colony measurements and cell number / colony calculations indicated that these colonies accumulated cells exceeding 10 mass doublings. According to one ‘stem cell’ model of tumor growth (Mackillop et al, JNCI 70: 9-16, 1983), such prolific colony growth is attributed to the self-renewal of cancer ‘stem cells’, whereas limited colony growth is achieved by partially differentiated cells. To assess whether prolific colonies form tumors in vivo, 10 single colonies derived from each colon cancer cell line were implanted into each of 10 athymic nu/nu mice. Testing of 5 tumor models completed to date shows that 75 – 100% (86% overall) of these colonies form tumor xenografts (median times to 700 mg tumor formation ranged from 40 to 72 days). Subsequent in vivo passage of 3 tumor xenografts per cell line resulted in tumor formation in 85 – 92% of host animals (median times ranging from 29 to 67 days). Thus, despite extensive in vitro propagation, a subset of individual cells in each cell line retained the functional capacity to initiate and sustain cancer cell growth in vitro and in vivo. Single cell cloning in soft agar provides an effective means to isolate individual colonies formed by cancer ‘stem cells’ and to propagate human tumor xenografts with modest numbers of tumor cells, host mice, and tumor formation times. The cell surface marker profile and drug sensitivity of prolific colonies are currently under investigation. (Funded in part by NCI Contract No. HHSN261200800001E) 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 5202. doi:10.1158/1538-7445.AM2011-5202

Abstract 3372: Analysis of putative tumor stem cell markers in the NCI-60 tumor cell line panel

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Tumor stem cells or cancer initiating cells are tumor cells that can regenerate a recurring tumor or a metastasis. A variety of cell surface and other markers have been associated with identification and characterization of tumor stem cells. We investigated the expression of a number of these putative stem cell markers (CD15, CD24, CD44, CD133, CD166, CD326, PgP, and aldehyde dehydrogenase) in the NCI60 tumor panel. A spectrally compatible set of labeled antibodies was designed to enable simultaneous evaluation of six markers (CD44, CD24, CD133, CD166, CD326 (EpCAM), and PgP, and forward (FSC) and side scatter (SSC). Marker expression patterns were analyzed by multiple component analysis and correlated with clonogeneic potential of cell lines (2D colony formation assay, anchorage independent colony formation in soft agar assay). Additionally sphere formation in suspension culture and the capacity of cell lines to form tumors in vivo upon injection with matrigel was assessed for colon cancer cell lines. Analysis of expression of single markers and co-expression of up to 6 markers demonstrated that all stem cell markers investigated are expressed in cell lines of the NCI-60 panel. Patterns of co-expression of these markers as defined by multiple component analysis tracked primarily with tumor type, but individual patterns were also observed to occur in multiple tumor types. In the breast cancer panel the patterns of co-expression segregated with the known molecular sub-types (basal, luminal, normal-like) of the tumor cell lines. Cells from colonosphere cultures generated from the colon tumor panel using serum-free, growth factor supplemented medium showed subtly different co-expression patterns. Both sphere-derived and bulk cultured colon tumor cells were highly tumorigenic in NOD/SCID mice and tumor formation was observed following inoculation of 10-100 cells. Only two out of seven of the colonosphere derived cell populations had enhanced tumor initiating ability, but did not share differences in stem cell marker display with respect to the unselected bulk cultures. Furthermore, marker expression patterns did not significantly correlate with in vitro colony formation (2D culture, soft agar). Our results demonstrate a high degree of complexity and heterogeneity in tumor stem cell marker display that can be influenced by culture conditions and relates to the histological classification of tumors rather than to the clonogenic potential of cells in cell lines of the NCI60 panel. 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 3372.