Self-renewing Tumor-initiating Cells Research Articles

Abstract 3582: Drugs targeting ovarian cancer tumor-initiating cells enhance oxidative stress and prevent disease recurrence

Abstract Ovarian cancer (OC) is a global health burden with the poorest 5-year survival rate of the gynecological malignancies. Disease recurrence is the major cause of morbidity and mortality of OC, likely driven by the survival of chemoresistant, self-renewing tumor-initiating cells (TICs). The TIC population in OC acquires features to survive cellular stress and avoid apoptosis but these can also be targeted as vulnerabilities specific to the TIC population of OC to prevent recurrence. To reveal these vulnerabilities, a high-throughput drug screen was performed to identify drugs that showed increased efficacy against OC cells grown in TIC-enriching spheroid (TES) conditions compared to adherently grown cells. Four drugs were selected for further investigation. Disulfiram, Bardoxolone Methyl, Elesclomol and Salinomycin were tested in vitro to confirm the effects on viability of OC cells in adherent or TES conditions. The drugs' efficacy against sphere formation and expression of markers of stemness as high ALDH activity and CD133 expression (ALDH+CD133+) as single agents or in combination with carboplatin was also investigated in vitro. Disulfiram showed the greatest efficacy against spheroid viability and decreased the ALDH+CD133+ population of several OC cell lines. This effect was enhanced when combined with carboplatin. Bardoxolone Methyl, Elesclomol and Salinomycin showed similar efficacy against cell viability in OC cells grown adherently or in TES conditions; Elesclomol significantly inhibited the ALDH+CD133+ population of the OC cells which was again enhanced in combination with carboplatin. RNAseq analysis revealed a strong enrichment of genes involved in oxidative phosphorylation and reactive oxygen species (ROS) pathways in TES conditions compared to adherent conditions. The drugs were investigated for their ability to promote oxidative stress and OC cells were measured for accumulation of intracellular ROS and mitochondrial superoxide after exposure to the drugs. OC cells grown in TES conditions had higher basal intracellular ROS levels than adherently grown cells and the level of ROS was significantly enhanced in TES cells treated with Elesclomol or Disulfiram but no differences were seen in adherently grown cells. Salinomycin and Bardoxolone Methyl increased mitochondrial superoxide accumulation in some OC cell lines under TES conditions. In a model of OC relapse in vitro, Disulfiram and Elesclomol following carboplatin treatment significantly increased cell death compared to carboplatin alone. Disulfiram and Salinomycin were incorporated into a mouse model to test their ability to prevent OC relapse after chemotherapy in vivo. These results demonstrate that targeting key pathways mediating increased oxidative stress in TICs can eliminate this population and provide a means to prevent OC recurrence. Citation Format: Brittney S. Harrington, Christina M. Annunziata. Drugs targeting ovarian cancer tumor-initiating cells enhance oxidative stress and prevent disease recurrence [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3582.

A perivascular niche in the bone marrow hosts quiescent and proliferating tumorigenic colorectal cancer cells

Disseminated tumor cells (dTCs) can frequently be detected in the bone marrow (BM) of colorectal cancer (CRC) patients, raising the possibility that the BM serves as a reservoir for metastatic tumor cells. Identification of dTCs in BM aspirates harbors the potential of assessing therapeutic outcome and directing therapy intensity with limited risk and effort. Still, the functional and prognostic relevance of dTCs is not fully established. We have previously shown that CRC cell clones can be traced to the BM of mice carrying patient-derived xenografts. However, cellular interactions, proliferative state and tumorigenicity of dTCs remain largely unknown. Here, we applied a coculture system modeling the microvascular niche and used immunofluorescence imaging of the murine BM to show that primary CRC cells migrate toward endothelial tubes. dTCs in the BM were rare, but detectable in mice with xenografts from most patient samples (8/10) predominantly at perivascular sites. Comparable to primary tumors, a substantial fraction of proliferating dTCs was detected in the BM. However, most dTCs were found as isolated cells, indicating that dividing dTCs rather separate than aggregate to metastatic clones-a phenomenon frequently observed in the microvascular niche model. Clonal tracking identified subsets of self-renewing tumor-initiating cells in the BM that formed tumors out of BM transplants, including one subset that did not drive primary tumor growth. Our results indicate an important role of the perivascular BM niche for CRC cell dissemination and show that dTCs can be a potential source for tumor relapse and tumor heterogeneity.

Nitroprusside induces melanoma ferroptosis with serum supplementation and prolongs survival under serum depletion or hypoxia

BackgroundWhen proliferating tumor cells expand to areas distant from vascular sites, poor diffusion of oxygen and nutrients occur, generating a restrictive hypoxic gradient in which susceptible tumor cells die. The heterogeneous population surviving hypoxia and metabolic starvation include de-differentiated cancer stem cells (CSC), capable of self-renewing tumor-initiating cells (TICs), or those that divide asymmetrically to produce non-tumor-initiating differentiated (NTI-D) cell progeny. Under such restrictive conditions, both populations slowly proliferate, entering quiescence or senescence, when exiting from cell cycle progression. This may drive chemoresistance and tumor recurrence, since most anti-cancer treatments target rapidly proliferating cells. PurposeSince persistent or additional stress may increase NTI-D cells conversion to TICs, we investigated whether nutrient depletion or hypoxia influence expression of tyrosinase, a crucial enzyme for melanin synthesis, and B16 melanoma survival, when exposed to iron-dependent cell death oxidative stress produced by the Fenton reaction, resembling ferroptosis. Results-a) proliferating B16 melanoma with 10% serum-supplementation (10%S) normoxically express hypoxia inducible factor 1α (HIF1α) but lose tyrosinase, in contrast to those transiently exposed to (SF) serum-free medium, in which both HIF1α and tyrosinase are co-expressed; b) in contrast to the resistance to SNP toxicity in (SF) cells with higher tyrosinase expression, those in (10%S) are killed by iron from nitroprusside/ferricyanide (SNP) irrespective of exogenous H2O2, in a reaction antagonized by the anti-oxidant and MEK inhibitor UO126; c) Moreover, under transient serum depletion, SNP cooperates with hypoxia (1.5% oxygen), prolonging B16 melanoma (SF) survival; d) the hypoxia mimetic CoCl2 inhibits proliferation-associated cyclin A, irrespective of SNP, in (10%S) cells or in transiently serum-depleted (SF) cells. However, only in the latter cells, CoCl2 but not SNP, induce loss of HIF1α and apoptosis-associated PARP cleavage; e) longer term adaptation to survive serum depletion, generates (SS) cells resistant to SNP toxicity, which aerobically co-express HIF1α and tyrosinase. In SS B16 melanoma, exogenous non-toxic 100 μM H2O2 super-induces the ratio of tyrosinase to HIF1α. However, co-treatment of SS-B16 cells with SNP plus exogenous H2O2, partly increases PARP cleavage by reciprocally decreasing tyrosinase expression. SIGNIFICANCE.- These results suggest that a phenotypic plasticity in response to depletion of nutrients and/or oxygen, helps decide whether melanoma cells undergo either death by ferroptosis, or resistance to it, when challenged by the same exogenous oxidative stress (iron ± H2O2).

Induced-Decay of Glycine Decarboxylase Transcripts as an Anticancer Therapeutic Strategy for Non-Small-Cell Lung Carcinoma.

Self-renewing tumor-initiating cells (TICs) are thought to be responsible for tumor recurrence and chemo-resistance. Glycine decarboxylase, encoded by the GLDC gene, is reported to be overexpressed in TIC-enriched primary non-small-cell lung carcinoma (NSCLC). GLDC is a component of the mitochondrial glycine cleavage system, and its high expression is required for growth and tumorigenic capacity. Currently, there are no therapeutic agents against GLDC. As a therapeutic strategy, we have designed and tested splicing-modulating steric hindrance antisense oligonucleotides (shAONs) that efficiently induce exon skipping (half maximal inhibitory concentration [IC50] at 3.5–7 nM), disrupt the open reading frame (ORF) of GLDC transcript (predisposing it for nonsense-mediated decay), halt cell proliferation, and prevent colony formation in both A549 cells and TIC-enriched NSCLC tumor sphere cells (TS32). One candidate shAON causes 60% inhibition of tumor growth in mice transplanted with TS32. Thus, our shAONs candidates can effectively inhibit the expression of NSCLC-associated metabolic enzyme GLDC and may have promising therapeutic implications.

BMI-1 Targeting Interferes with Patient-Derived Tumor-Initiating Cell Survival and Tumor Growth in Prostate Cancer.

Current prostate cancer management calls for identifying novel and more effective therapies. Self-renewing tumor-initiating cells (TICs) hold intrinsic therapy resistance and account for tumor relapse and progression. As BMI-1 regulates stem cell self-renewal, impairing BMI-1 function for TIC-tailored therapies appears to be a promising approach. We have previously developed a combined immunophenotypic and time-of-adherence assay to identify CD49bhiCD29hiCD44hi cells as human prostate TICs. We utilized this assay with patient-derived prostate cancer cells and xenograft models to characterize the effects of pharmacologic inhibitors of BMI-1. We demonstrate that in cell lines and patient-derived TICs, BMI-1 expression is upregulated and associated with stem cell-like traits. From a screened library, we identified a number of post-transcriptional small molecules that target BMI-1 in prostate TICs. Pharmacologic inhibition of BMI-1 in patient-derived cells significantly decreased colony formation in vitro and attenuated tumor initiation in vivo, thereby functionally diminishing the frequency of TICs, particularly in cells resistant to proliferation- and androgen receptor-directed therapies, without toxic effects on normal tissues. Our data offer a paradigm for targeting TICs and support the development of BMI-1-targeting therapy for a more effective prostate cancer treatment. Clin Cancer Res; 22(24); 6176-91. ©2016 AACR.

High expression of TROP2 characterizes different cell subpopulations in androgen-sensitive and androgen-independent prostate cancer cells.

Progression of castration-resistant tumors is frequent in prostate cancer. Current systemic treatments for castration-resistant prostate cancer only produce modest increases in survival time and self-renewing Tumor-Initiating Cells (TICs) are suspected to play an important role in resistance to these treatments. However it remains unclear whether the same TICs display both chemo-resistance and self-renewing abilities throughout progression from early stage lesions to late, castration resistant tumors. Here, we found that treatment of mice bearing LNCaP-derived xenograft tumors with cytotoxic (docetaxel) and anti-androgen (flutamide) compounds enriched for cells that express TROP2, a putative TIC marker. Consistent with a tumor-initiating role, TROP2high cells from androgen-sensitive prostate cancer cell lines displayed an enhanced ability to re-grow in culture following treatment with taxane-based chemotherapy with or without androgen blockade. TROP2 down-regulation in these cells reduced their ability to recur after treatment with docetaxel, in the presence or absence of flutamide. Accordingly, in silico analysis of published clinical data revealed that prostate cancer patients with poor prognosis exhibit significantly elevated TROP2 expression level compared to low-risk patients, particularly in the case of patients diagnosed with early stage tumors. In contrast, in androgen-independent prostate cancer cell lines, TROP2high cells did not exhibit a differential treatment response but were characterized by their high self-renewal ability. Based on these findings we propose that high TROP2 expression identifies distinct cell sub-populations in androgen-sensitive and androgen-independent prostate tumors and that it may be a predictive biomarker for prostate cancer treatment response in androgen-sensitive tumors.

Collaboration of 3D context and extracellular matrix in the development of glioma stemness in a 3D model

A hierarchy of cellular stemness exists in certain cancers, and any successful strategy to treat such cancers would have to eliminate the self-renewing tumor-initiating cells at the apex of the hierarchy. The cellular microenvironment, in particular the extracellular matrix (ECM), is believed to have a role in regulating stemness. In this work, U251 glioblastoma cells are cultured on electrospun polystyrene (ESPS) scaffolds coated with an array of 7 laminin isoforms to provide a 3D model for stem cell-related genes and proteins expression studies. We observed collaboration between 3D context and laminins in promoting glioma stemness. Depending on the laminin isoform presented, U251 cells cultured on ESPS scaffolds (3D) exhibited increased expression of stemness markers compared to those cultured on tissue culture polystyrene (2D). Our results indicate the influence of 3D (versus 2D) context on integrin expression, specifically, the upregulation of the laminin-binding integrins alpha 6 and beta 4. By a colony forming assay, we showed enhanced clonogenicity of cells grown on ESPS scaffolds in collaboration with laminins 411, 421, 511 and 521. Evaluation of patient glioma databases demonstrated significant enrichment of integrin and ECM pathway networks in tumors of worse prognosis, consistent with our observations. The present results demonstrate how 3D versus 2D context profoundly affects ECM signaling, leading to stemness.

Aptamer Identification of Brain Tumor–Initiating Cells

Glioblastomas display cellular hierarchies with self-renewing tumor-initiating cells (TIC), also known as cancer stem cells, at the apex. Although the TIC hypothesis remains controversial and the functional assays to define the TIC phenotype are evolving, we and others have shown that TICs may contribute to therapeutic resistance, tumor spread, and angiogenesis. The identification of TICs has been informed by the use of markers characterized in normal stem cells, but this approach has an inherent limitation to selectively identify TICs. To develop reagents that enrich TICs but not matched non-TICs or tissue-specific stem cells, we adopted Cell-Systematic Evolution of Ligands by Exponential Enrichment (Cell-SELEX) to identify glioblastoma TIC-specific nucleic acid probes-aptamers-that specifically bind TICs. In this study, using Cell-SELEX with positive selection for TICs and negative selection for non-TICs and human neural progenitor cells, we identified TIC aptamers that specifically bind to TICs with excellent dissociation constants (Kd). These aptamers select and internalize into glioblastoma cells that self-renew, proliferate, and initiate tumors. As aptamers can be modified to deliver payloads, aptamers may represent novel agents that could selectively target or facilitate imaging of TICs.

Abstract 2295: Succession of transiently active TIC clones drives long-term human pancreatic cancer progression .

Abstract Despite accumulating evidence indicating a pivotal role of tumor-initiating cells (TIC) in tumor progression and metastases formation of pancreatic cancer (PC), very little is known about the clonal TIC dynamics within PC tumors in vivo. It is unclear whether the high aggressiveness of PC is driven by a homogeneous population of self-renewing TIC or whether distinct classes or activation states of TIC drive long-term disease progression. To address this we tracked the clonal contribution of individual TIC to tumor formation in vivo. Serum-free adherent cultures were established allowing enrichment of PC TIC without restriction to a certain phenotype. These cultures grow as 3-dimensional epithelial colonies with tight cell-to-cell contacts und reliably form subcutaneous tumors in NSG mice. To induce differentiation of TIC, FBS was added and FGF2, FGF10 and Nodal were withdrawn. Under these conditions, irregular shaped cells form a monolayer and down regulate markers previously described for TIC or normal pancreatic progenitors. Strikingly, despite profound changes in morphology and marker expression tumor initiation and self-renewal of TIC in serial transplantation were unchanged. Moreover, sorted CD133+ and CD133− cells equally efficient formed tumors containing similar proportions of CD133+ in vivo. Together these data indicate an unexpected phenotypic plasticity of TIC in PC. To determine the clonal kinetics of individual self-renewing TIC in vivo, early passage serum-free cultures from 3 patients were transduced with a lentiviral vector (LV) and serially transplanted. LV stably integrate into the host cell genome resulting in TIC clone specific fusion sequences which were identified by highly sensitive LAM-PCR and high throughput sequencing. In primary mice, 0.003-0.113% of all transduced cells contributed actively to tumor formation. Unexpectedly, in subsequent generations tumor formation was mainly driven by distinct TIC clones not detectable in earlier but recruited to tumor formation in later generations. Moreover, individual primary xenografts generated pairs of secondary tumors with very little overlap between clonal compositions. Mathematical modeling indicates strong changes in proliferative activity of individual, otherwise homogenous TIC which predominantly produce non-tumorigenic progeny with very limited self-renewal. These data indicate that long-term progression of PC is driven by a succession of transiently active TIC generating tumor cells in temporally restricted bursts. The recruitment of inactive TIC clones to tumor formation after serial transplantation points to a context-dependent regulation of TIC activity within the growing tumor. Further understanding the mechanisms regulating the balance between activated and resting states of TIC may allow developing specific treatment strategies targeting this most relevant cell population in PC. Citation Format: Felix Oppel, Claudia R. Ball, Christopher M. Hoffmann, Sebastian M. Dieter, Taronish D. Dubash, Moritz Koch, Jürgen Weitz, Frank Bergmann, Manfred Schmidt, Ulrich Abel, Christof von Kalle, Hanno Glimm. Succession of transiently active TIC clones drives long-term human pancreatic cancer progression . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2295. doi:10.1158/1538-7445.AM2013-2295

T-53 Mechanic-based reprogramming of hepatic self-renewing tumor-initiating cells through DNMT1-inhibition

T-53 Mechanic-based reprogramming of hepatic self-renewing tumor-initiating cells through DNMT1-inhibition

Abstract 4312: Identification of putative stem cell markers of CD133 and CD44 in human ovarian cancer specimens and ovarian cancer cell lines

Abstract Objective: Ovarian cancer may arise from a subpopulation of CD133+ 44+ cells, which are self-renewing tumor-initiating cells. Materials and Methods: We investigated the tumorigenicity of the cell line A2780 which is CD133high44low, the cell line A2780 with siRNA against CD133 (CD133low44low), and human ovarian cancer cells from ascites, from primary ovarian cancer, and from metastatic ovarian cancer (CD133 high/CD44high) harvested from patients at the time of surgical cytoreduction in ultra low attachment plates in serum free medium. Spheroids from each of these sources were used to test their tumorigenicity in SCID mice. Results: Cells from A2780 cell line (CD133 high/CD44low) formed tumors in SCID mice in 2 months but silencing CD133 with siRNA CD133 (CD133 low/CD44low) inhibited tumorigenicity in SCID mice and retarded but did not prevent spheroid formation. Spheroid cultures of ascites, primary ovary, and metastasis (CD133 high/CD44high) are capable of inducing primary ovarian tumors and metastatic tumors in SCID mice in 2 months compared to 4-6 months in sorted, non-spheroid cells. Conclusions: The data suggest that CD133 expression is necessary for tumorigenesis in the A2780 cell line but not for spheroid formation. Our data also suggests that spheroids which are CD133 high/CD44high from human tumor recapitulate ovarian cancer in women. These novel in vivo and vitro models may offer useful tools to develop therapies targeted to ovarian cancer. 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 4312.

Abstract 9: Distinct types of human colon cancer initiating cells contribute to primary tumor and metastasis formation

Abstract Strong evidence indicates that rare tumor initiating cells (TICs) within tumors play a pivotal role in initiation and maintenance of genetically and morphologically heterogeneous human colon cancers. Self-renewing TICs form serially transplantable tumors that resemble the original patient tumor in immunodeficient mice. Up to now, it is unknown whether TICs represent a homogeneous population with identical potential to self-renew and metastasize, or if they are organized in a hierarchically structured cancer stem cell compartment. Here we demonstrate an unexpected cellular heterogeneity within the TIC compartment by lentiviral marking and in vivo clonal tracking of individual, highly enriched primary human colon cancer initiating cells. TICs derived from different primary human colon tumors or metastases were enriched as tumor spheres under serum-free culture conditions. Frequency of cells capable of forming new tumor spheres after replating ranged from 4%-60% depending on the individual patient. In vivo self-renewal and metastatic capacity of single primary colon TICs in serially transplanted tumors and associated metastases were analyzed by highly sensitive tracking of individual lentivirally marked clones. Primary tumor cells were serially transplanted under the renal capsule of immunodeficient NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (IL2RGnull) mice. Clonal contribution of TICs individually marked by unique lentiviral integration sites was detected by linear amplification-mediated PCR (LAM-PCR) followed by next generation high throughput sequencing. Three distinct types of TICs contributed to tumor formation: 1) previously unnoticed tumor transient amplifying cells (T-TACs) with limited or no self-renewal capacity contributing only to tumor formation in primary mice, 2) rare extensively self-renewing long-term tumor initiating cells (LT-TICs) that maintain tumor formation after serial transplantation and 3) ‘dormant’ TICs that become active in secondary or tertiary mice. Only a subfraction of all TIC clones metastasized into bone marrow or liver, indicating that tumor initiation, self-renewal as well as metastasis forming potential are independently regulated in TICs. Our results allow for the first time detailed insights into the clonal heterogeneity within the human colon cancer initiating cell compartment and identify three distinct types of TICs with different self-renewal and metastasis-forming potential. They indicate that tumor initiation, self-renewal and metastasis formation of TICs are independently regulated. Understanding the regulatory mechanisms underlying self-renewal capacity and metastasis formation in LT-TICs will have important implications for future curative treatment strategies in human colon cancer. 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 9.

A Hierarchy of Self-Renewing Tumor-Initiating Cell Types in Glioblastoma

The neural stem cell marker CD133 is reported to identify cells within glioblastoma (GBM) that can initiate neurosphere growth and tumor formation; however, instances of CD133(-) cells exhibiting similar properties have also been reported. Here, we show that some PTEN-deficient GBM tumors produce a series of CD133(+) and CD133(-) self-renewing tumor-initiating cell types and provide evidence that these cell types constitute a lineage hierarchy. Our results show that the capacities for self-renewal and tumor initiation in GBM need not be restricted to a uniform population of stemlike cells, but can be shared by a lineage of self-renewing cell types expressing a range of markers of forebrain lineage.

P21 CIP1 attenuates Ras- and c-Myc-dependent breast tumor epithelial mesenchymal transition and cancer stem cell-like gene expression in vivo

p21(CIP1/WAF1) is a downstream effector of tumor suppressors and functions as a cyclin-dependent kinase inhibitor to block cellular proliferation. Breast tumors may derive from self-renewing tumor-initiating cells (BT-ICs), which contribute to tumor progression, recurrence, and therapy resistance. The role of p21(CIP1) in regulating features of tumor stem cells in vivo is unknown. Herein, deletion of p21(CIP1), which enhanced the rate of tumorigenesis induced by mammary-targeted Ha-Ras or c-Myc, enhanced gene expression profiles and immunohistochemical features of epithelial mesenchymal transition (EMT) and putative cancer stem cells in vivo. Silencing of p21(CIP1) enhanced, and expression of p21(CIP1) repressed, features of EMT in transformed immortal human MEC lines. p21(CIP1) attenuated oncogene-induced BT-IC and mammosphere formation. Thus, the in vitro cell culture assays reflect the changes observed in vivo in transgenic mice. These findings establish a link between the loss of p21(CIP1) and the acquisition of breast cancer EMT and stem cell properties in vivo.

The role of PTEN/Akt/PI3K signaling in the maintenance and viability of prostate cancer stem-like cell populations

Characterization of the molecular pathways that are required for the viability and maintenance of self-renewing tumor-initiating cells may ultimately lead to improved therapies for cancer. In this study, we show that a CD133(+)/CD44(+) population of cells enriched in prostate cancer progenitors (PCaPs) has tumor-initiating potential and that these progenitors can be expanded under nonadherent, serum-free, sphere-forming conditions. Cells grown under these conditions have increased in vitro clonogenic and in vivo tumorigenic potential. mRNA expression analysis of cells grown under sphere-forming conditions, compared with long-term monolayer cultures, revealed preferential activation of the PI3K/AKT signaling pathway. PI3K p110alpha and beta-protein levels were higher in cells grown under sphere-forming conditions, and phosphatase and tensin homolog (PTEN) knockdown by shRNA led to an increase in sphere formation as well as increased clonogenic and tumorigenic potential. Similarly, shRNA knockdown of FoxO3a led to an increase in tumorigenic potential. Consistent with these results, inhibition of PI3K activity by the dual PI3K/mTOR inhibitor NVP-BEZ235 led to growth inhibition of PCaPs. Taken together, our data strongly suggest that the PTEN/PI3K/Akt pathways are critical for prostate cancer stem-like cell maintenance and that targeting PI3K signaling may be beneficial in prostate cancer treatment by eliminating prostate cancer stem-like cells.

PC3 Human Prostate Carcinoma Cell Holoclones Contain Self-renewing Tumor-Initiating Cells

Primary keratinocytes exhibit three typical clonal morphologies represented by holoclones, meroclones, and paraclones, with holoclones containing self-renewing stem cells, and meroclones and paraclones containing more mature and differentiated cells. Interestingly, long-term-cultured human epithelial cancer cells in clonal cultures also form holoclones, meroclones, and paraclones, and tumor cell holoclones have been hypothesized to harbor stem-like cells or cancer stem cells. However, the key question of whether tumor cell holoclones genuinely contain tumor-initiating cells has not been directly addressed. Here, using PC3 human prostate carcinoma cells as a model, we provide direct experimental evidence that tumor cell holoclones contain stem-like cells that can initiate serially transplantable tumors. Importantly, holoclones derived from either cultured PC3 cells or holoclone-initiated tumors can be serially passaged and regenerate all three types of clones. In contrast, meroclones and paraclones cannot be continuously propagated and fail to initiate tumor development. Phenotypic characterizations reveal high levels of CD44, alpha(2)beta(1) integrin, and beta-catenin expression in holoclones, whereas meroclones and paraclones show markedly reduced expression of these stem cell markers. The present results have important implications in understanding morphologic heterogeneities and tumorigenic hierarchies in human epithelial cancer cells.

Let-7 Regulates Self Renewal and Tumorigenicity of Breast Cancer Cells

Cancers may arise from rare self-renewing tumor-initiating cells (T-IC). However, how T-IC self renewal, multipotent differentiation, and tumorigenicity are maintained remains obscure. Because miRNAs can regulate cell-fate decisions, we compared miRNA expression in self-renewing and differentiated cells from breast cancer lines and in breast T-IC (BT-IC) and non-BT-IC from 1 degrees breast cancers. let-7 miRNAs were markedly reduced in BT-IC and increased with differentiation. Infecting BT-IC with let-7-lentivirus reduced proliferation, mammosphere formation, and the proportion of undifferentiated cells in vitro and tumor formation and metastasis in NOD/SCID mice, while antagonizing let-7 by antisense oligonucleotides enhanced in vitro self renewal of non-T-IC. Increased let-7 paralleled reduced H-RAS and HMGA2, known let-7 targets. Silencing H-RAS in a BT-IC-enriched cell line reduced self renewal but had no effect on differentiation, while silencing HMGA2 enhanced differentiation but did not affect self renewal. Therefore let-7 regulates multiple BT-IC stem cell-like properties by silencing more than one target.