Glycolipid GD3 and GD3 synthase are key drivers for glioblastoma stem cells and tumorigenicity

The cancer stem cells (CSCs) of glioblastoma multiforme (GBM), a grade IV astrocytoma, have been enriched by the expressed marker CD133. However, recent studies have shown that CD133⁻ cells also possess tumor-initiating potential. By analysis of gangliosides on various cells, we show that ganglioside D3 (GD3) is overexpressed on eight neurospheres and tumor cells; in combination with CD133, the sorted cells exhibit a higher expression of stemness genes and self-renewal potential; and as few as six cells will form neurospheres and 20–30 cells will grow tumor in mice. Furthermore, GD3 synthase (GD3S) is increased in neurospheres and human GBM tissues, but not in normal brain tissues, and suppression of GD3S results in decreased GBM stem cell (GSC)-associated properties. In addition, a GD3 antibody is shown to induce complement-dependent cytotoxicity against cells expressing GD3 and inhibition of GBM tumor growth in vivo. Our results demonstrate that GD3 and GD3S are highly expressed in GSCs, play a key role in glioblastoma tumorigenicity, and are potential therapeutic targets against GBM.Glioblastoma multiforme (GBM) is extremely infiltrative and difficult to treat, and most patients develop recurrence after therapy. Over the past decade, many studies have suggested that bulk GBM tumors harbor cancer stem cells (CSCs) (1, 2), a distinct subpopulation of cancer cells that are able to initiate new tumors efficiently, have long-term self-renewal capacity, and survive better against chemo- or radiotherapy (2–4). CD133 has become a widely used marker for the enrichment of GBM CSCs (GSCs) and other tumor types (5–10). However, recent studies have shown that CD133 is not specific for GSCs because CD133⁻ cells also possess tumor-initiating potential (11–13), indicating the need to identify more specific and exclusive markers for GSCs to facilitate our understanding of GSCs and therapeutic development against GBM. Several reports have proposed L1CAM, A2B5, integrin α6, MET, and CD15 as markers for GSCs (14–18). However, none of these protein markers could be used specifically to identify GSCs, and no study was reported with respect to glycans as potential markers, although glycan biosynthesis involves multiple genes and it is possible to create different structures in cancer progression. It is noted that ganglioside D2 (GD2) and ganglioside D3 (GD3) were found on the surface of neural stem cells (NSCs) and that stage-specific embryonic antigen 3 (SSEA3) and SSEA4 were found on embryonic stem cells and cancer cells (19–21), but there is no glycan marker found on the surface of GSCs.Gangliosides are sialic acid-containing glycosphingolipids (GSLs) that are most abundant in the nervous system (22). The expression levels and patterns of gangliosides during brain development shift from simple gangliosides, such as GM3 and GD3, to complex gangliosides, such as GM1, GD1a, GD1b, and GT1b (23, 24). Moreover, several unique ganglioside markers, including SSEA3, SSEA4, GD2, and GD3, have been identified in stem cells (19). GD3, a b-series ganglioside containing two sialic acids, is highly expressed in mouse and human embryonic NSCs (20, 25). In cancers, GD3 is highly accumulated in human primary melanoma tissues as well as in established melanoma cell lines (26), whereas human normal melanocytes express no or minimal levels of GD3 (27). Moreover, malignant gliomas contain higher levels of GD3, and its expression correlates with the degree of malignancy (28). GD3 is produced from the precursor GM3 by the activity of GD3 synthase (GD3S), which mediates the properties of CSCs through the c-MET signaling pathway and correlates with poor prognosis in triple-negative human breast tumors (29). These findings suggest that GD3 may play an important role in the transformation of normal cells into tumors, and imply that GD3 could be a cell surface marker for GSCs.This study was designed to identify glycan markers for the enrichment of GBM stem cells and then uses these enriched GBM stem cells to characterize tumorigenicity, their association with clinical GBM specimens, and their regulation in tumor progression. The results showed that GD2 and GD3 were positively stained on GBM neurospheres. We found that cells with high GD3 expression display functional characteristics of GSCs. Suppression of GD3S, a critical enzyme for GD3 synthesis, impeded neurosphere formation and tumor initiation. The expression of GD3S correlated with the grades of astrocytomas and mediated self-renewal through c-Met activation. Furthermore, a GD3 antibody was found to eliminate the GD3⁺ cells through complement-dependent cytotoxicity (CDC) in vitro and to suppress tumor growth in mice. These results suggest that GD3 could be a significant biomarker for GSCs, that CD3 could be combined with CD133 for the enrichment of GSCs, and that both GD3 and GD3S could be targets for the development of new therapies against GBM.     

Potentiation of cytotoxic chemotherapy by growth hormone-releasing hormone agonists

The dismal prognosis of malignant brain tumors drives the development of new treatment modalities. In view of the multiple activities of growth hormone-releasing hormone (GHRH), we hypothesized that pretreatment with a GHRH agonist, JI-34, might increase the susceptibility of U-87 MG glioblastoma multiforme (GBM) cells to subsequent treatment with the cytotoxic drug, doxorubicin (DOX). This concept was corroborated by our findings, in vivo, showing that the combination of the GHRH agonist, JI-34, and DOX inhibited the growth of GBM tumors, transplanted into nude mice, more than DOX alone. In vitro, the pretreatment of GBM cells with JI-34 potentiated inhibitory effects of DOX on cell proliferation, diminished cell size and viability, and promoted apoptotic processes, as shown by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide proliferation assay, ApoLive-Glo multiplex assay, and cell volumetric assay. Proteomic studies further revealed that the pretreatment with GHRH agonist evoked differentiation decreasing the expression of the neuroectodermal stem cell antigen, nestin, and up-regulating the glial maturation marker, GFAP. The GHRH agonist also reduced the release of humoral regulators of glial growth, such as FGF basic and TGFβ. Proteomic and gene-expression (RT-PCR) studies confirmed the strong proapoptotic activity (increase in p53, decrease in v-myc and Bcl-2) and anti-invasive potential (decrease in integrin α3) of the combination of GHRH agonist and DOX. These findings indicate that the GHRH agonists can potentiate the anticancer activity of the traditional chemotherapeutic drug, DOX, by multiple mechanisms including the induction of differentiation of cancer cells.Glioblastoma multiforme (GBM) is one of the most aggressive human cancers, and the afflicted patients inevitably succumb. The dismal outcome of this malignancy demands great efforts to find improved methods of treatment (1). Many compounds have been synthesized in our laboratory in the past few years that have proven to be effective against diverse malignant tumors (2–14). These are peptide analogs of hypothalamic hormones: luteinizing hormone-releasing hormone (LHRH), growth hormone-releasing hormone (GHRH), somatostatin, and analogs of other neuropeptides such as bombesin and gastrin-releasing peptide. The receptors for these peptides have been found to be widely distributed in the human body, including in many types of cancers (2–14). The regulatory functions of these hypothalamic hormones and other neuropeptides are not confined to the hypothalamo–hypophyseal system or, even more broadly, to the central nervous system (CNS). In particular, GHRH can induce the differentiation of ovarian granulosa cells and other cells in the reproductive system and function as a growth factor in various normal tissues, benign tumors, and malignancies (2–4, 6, 11, 14–18). Previously, we also reported that antagonistic cytototoxic derivatives of some of these neuropeptides are able to inhibit the growth of several malignant cell lines (2–14).Our earlier studies showed that treatment with antagonists of LHRH or GHRH rarely effects complete regression of glioblastoma-derived tumors (5, 7, 10, 11). Previous studies also suggested that growth factors such as EGF or agonistic analogs of LHRH serving as carriers for cytotoxic analogs and functioning as growth factors may sensitize cancer cells to cytotoxic treatments (10, 19) through the activation of maturation processes. We therefore hypothesized that pretreatment with one of our GHRH agonists, such as JI-34 (20), which has shown effects on growth and differentiation in other cell lines (17, 18, 21, 22), might decrease the pluripotency and the adaptability of GBM cells and thereby increase their susceptibility to cytotoxic treatment.In vivo, tumor cells were implanted into athymic nude mice, tumor growth was recorded weekly, and final tumor mass was measured upon autopsy. In vitro, proliferation assays were used for the determination of neoplastic proliferation and cell growth. Changes in stem (nestin) and maturation (GFAP) antigen expression was evaluated with Western blot studies in vivo and with immunocytochemistry in vitro. The production of glial growth factors (FGF basic, TGFβ) was verified by ELISA. Further, using the Human Cancer Pathway Finder real-time quantitative PCR, numerous genes that play a role in the development of cancer were evaluated. We placed particular emphasis on the measurement of apoptosis, using the ApoLive-Glo Multiplex Assay kit and by detection of the expression of the proapoptotic p53 protein. This overall approach permitted the evaluation of the effect of GHRH agonist, JI-34, on the response to chemotherapy with doxorubicin.     

CD14-expressing cancer cells establish the inflammatory and proliferative tumor microenvironment in bladder cancer

Nonresolving chronic inflammation at the neoplastic site is consistently associated with promoting tumor progression and poor patient outcomes. However, many aspects behind the mechanisms that establish this tumor-promoting inflammatory microenvironment remain undefined. Using bladder cancer (BC) as a model, we found that CD14-high cancer cells express higher levels of numerous inflammation mediators and form larger tumors compared with CD14-low cells. CD14 antigen is a glycosyl-phosphatidylinositol (GPI)-linked glycoprotein and has been shown to be critically important in the signaling pathways of Toll-like receptor (TLR). CD14 expression in this BC subpopulation of cancer cells is required for increased cytokine production and increased tumor growth. Furthermore, tumors formed by CD14-high cells are more highly vascularized with higher myeloid cell infiltration. Inflammatory factors produced by CD14-high BC cells recruit and polarize monocytes and macrophages to acquire immune-suppressive characteristics. In contrast, CD14-low BC cells have a higher baseline cell division rate than CD14-high cells. Importantly, CD14-high cells produce factors that further increase the proliferation of CD14-low cells. Collectively, we demonstrate that CD14-high BC cells may orchestrate tumor-promoting inflammation and drive tumor cell proliferation to promote tumor growth.Solid tumors represent a complex mass of tissue composed of multiple distinct cell types (1, 2). Cells within the tumor produce a range of soluble factors to create a complex of signaling networks within the tumor microenvironment (3–7). One of the outcomes of this crosstalk is tumor-promoting inflammation (TPI) (8, 9). TPI can modulate the functions of tumor-infiltrating myeloid lineage cells including macrophages (10–12). Tumor-associated macrophages (TAMs) consistently display an alternatively activated phenotype (M2) commonly found in sites of wound healing (13–18). These macrophages promote tumor growth while suppressing the host immune response locally (19–22). Polarization and subversion of tumor-infiltrating macrophages is accomplished via immune mediators in the tumor microenvironment (23, 24). Adding to the complexity of solid tumors is the heterogeneity of the cancer cells (2). Tumor cells of varying differentiation states and different characteristics coexist within a tumor (25–29). However, the different roles of each tumor cell subset during cancer progression remain undefined.Bladder cancer (BC) represents a growing number of solid tumors characterized by the infiltration of a significant number of myeloid cells in the neoplastic lesion (30, 31). We have previously determined that keratin 14 (KRT14) expression marks the most primitive differentiation state in BC cells (32). KRT14 expression is significantly associated with poor overall patient survival. However, the mechanisms used by KRT14-expressing cells to promote tumor growth remain unclear. In the current study, we found that KRT14+ basal BC cells also express higher levels of CD14. Here, we investigate the strategies used by KRT14+ CD14-high BC cells to promote tumor growth.     

Efficient germ-line transmission obtained with transgene-free induced pluripotent stem cells

Induced pluripotent stem (iPS) cells hold great promise for regenerative medicine. To overcome potential problems associated with transgene insertions, efforts have been directed over the past several years to generate transgene-free iPS cells by using non-viral-vector approaches. To date, however, cells generated through such procedures have had problems producing reproductively competent animals, suggesting that their quality needed further improvement. Here we report the use of optimized assemblies of reprogramming factors and selection markers incorporated into single plasmids as nonintegrating episomes to generate germ-line–competent iPS cells. In particular, the pMaster12 episome can produce transgene-free iPS cells that, when grown in 2i medium, recapitulate good mouse ES cells, in terms of their competency for generating germ-line chimeras.Although induced pluripotent stem (iPS) cells hold enormous promise for cell-based therapies (1, 2), their use in humans is still problematic because of their potential to also do harm to the patient. High among these risk factors is their potential to induce cancer. For example, use of viral vectors to randomly integrate genes into somatic cells used for human gene therapy trials has resulted in the induction of cancer in the recipient patients (3). Also, random integration of the exogenous reprograming genes in iPS cells increases tumor formation, morbidity, and mortality in mice generated from these cells (4, 5). Therefore, the safety and quality of iPS cells are of critical importance to their anticipated use for human cell-based therapies. To circumvent some of these safety issues, safer methods, especially nonviral methods, for the introduction of the reprogramming factors into somatic cells are being developed. These methods have included the use of plasmids (6, 7), the piggyBac (PB) transposon (8, 9), nonintegrating episomes (10–13), protein transduction (14, 15), transfection of mRNA and microRNAs (16–18), and small molecule inhibitors (19). Although these approaches have yielded transgene-free iPS cells, the competency of the resulting iPS cells to contribute to a functional germ line has not been adequately demonstrated.     

Highly penetrative,drug-loaded nanocarriers improve treatment of glioblastoma

Current therapy for glioblastoma multiforme is insufficient, with nearly universal recurrence. Available drug therapies are unsuccessful because they fail to penetrate through the region of the brain containing tumor cells and they fail to kill the cells most responsible for tumor development and therapy resistance, brain cancer stem cells (BCSCs). To address these challenges, we combined two major advances in technology: (i) brain-penetrating polymeric nanoparticles that can be loaded with drugs and are optimized for intracranial convection-enhanced delivery and (ii) repurposed compounds, previously used in Food and Drug Administration-approved products, which were identified through library screening to target BCSCs. Using fluorescence imaging and positron emission tomography, we demonstrate that brain-penetrating nanoparticles can be delivered to large intracranial volumes in both rats and pigs. We identified several agents (from Food and Drug Administration-approved products) that potently inhibit proliferation and self-renewal of BCSCs. When loaded into brain-penetrating nanoparticles and administered by convection-enhanced delivery, one of these agents, dithiazanine iodide, significantly increased survival in rats bearing BCSC-derived xenografts. This unique approach to controlled delivery in the brain should have a significant impact on treatment of glioblastoma multiforme and suggests previously undescribed routes for drug and gene delivery to treat other diseases of the central nervous system.Of the ∼40,000 people diagnosed with primary brain tumors in the United States each year, an estimated 15,000 have glioblastoma multiforme (GBM), a World Health Organization grade IV malignant glioma (1). Despite considerable research efforts, the prognosis for GBM remains poor: median survival with standard-of-care therapy (surgery, systemic chemotherapy with temozolomide, and radiation) is 14.6 mo (2) and 5-y survival is 9.8% (3), with the vast majority of GBMs recurring within 2 cm of the original tumor focus (4). Histopathologically, GBM is characterized by its infiltrative nature and cellular heterogeneity, leading to a number of challenges that must be overcome by any presumptive therapy.The blood–brain barrier (BBB) is a major obstacle to treating GBM (5). It is estimated that over 98% of small-molecule drugs and ∼100% of large-molecule drugs or genes do not cross the BBB (6). Delivery of chemotherapeutics to the brain can be potentially achieved by using nanocarriers engineered for receptor-mediated transport across the BBB (7, 8), but the percentage of i.v. administered particles that enter the brain is low. It is not yet clear whether sufficient quantities of drug can be delivered by systemically administered nanoparticles to make this a useful method for treating tumors in the human brain. An alternate approach is to bypass the BBB: Clinical trials have demonstrated that the BBB can be bypassed with direct, locoregional delivery of therapeutic agents. For example, local implantation of a drug-loaded biodegradable polymer wafer (presently marketed as Gliadel), which slowly releases carmustine over a prolonged period, is a safe method for treating GBM. However, use of the Gliadel wafer results in only modest improvements in patient survival, typically 2 mo (9, 10). In prior work we showed that these wafers produce high interstitial drug concentrations in the tissue near the implant, but—because drugs move from the implant into the tissue by diffusion—penetration into tissue is limited to ∼1 mm, which could limit their efficacy (11, 12).We hypothesize that treatment of GBM can be improved by attention to three challenges: (i) enhancing the depth of penetration of locally delivered therapeutic agents, (ii) providing for long-term release of active agents, and (iii) delivering agents that are known to be effective against the cells that are most important in tumor recurrence. The first challenge can be addressed by convection-enhanced delivery (CED), in which agents are infused into the brain under a positive pressure gradient, creating bulk fluid movement in the brain interstitium (13). Recent clinical trials show that CED is safe and feasible (14–16), but CED alone is not sufficient to improve GBM treatment. For example, CED of a targeted toxin in aqueous suspension failed to show survival advantages over Gliadel wafers (14, 17). Although CED of drugs in solution results in increased penetration, most drugs have short half-lives in the brain and, as a result, they disappear soon after the infusion stops (17, 18). Loading of agents into nanocarriers—such as liposomes, micelles, dendrimers, or nanoparticles—can protect them from clearance. Significant progress has been made in CED of liposomes to the brain (19), although it is not clear that liposomes offer the advantage of long-term release. By contrast, CED of polymeric nanoparticles, such as nanoparticles made of poly(lactide-coglycolide) (PLGA), offers the possibility of controlled agent release. However, CED of PLGA nanoparticles, which are typically 100–200 nm in diameter, has been limited by the failure of particles to move by convection through the brain interstitial spaces (20–23), which are 38–64 nm in normal brain (24) and 7–100 nm in regions with tumor (25). Therefore, to overcome the first and second challenges, it is necessary to synthesize polymer nanocarriers that are much smaller than conventional particles and still capable of efficient drug loading and controlled release. We report here reliable methods for making PLGA nanoparticles with these characteristics.Drug developers have long been frustrated by the BBB, which severely limits the types of agents that can be tested for activity in the brain. We reasoned that creation of safe, versatile, brain-penetrating nanocarriers should enable direct testing of novel agents that address the complexity of GBM biology. For example, cells isolated from distinct regions of a given GBM bear grossly different expression signatures but seem to arise from a common progenitor (26): A small subpopulation of these progenitors drives tumor progression, promotes angiogenesis, and influences tumor cell migration (27–30). These cells have features of primitive neural stem cells and are called brain cancer stem cells (BCSCs) (29, 31–37). BCSCs, many of which are marked by CD133 (PROM1), are resistant to conventional drugs (28, 38), including carboplatin, cisplatin, paclitaxel, doxorubicin, vincristine, methotrexate, and temozolomide (39–42), as well as radiotherapy (29). These observations suggest that agents that affect BCSCs are more likely to lead to a cure for GBM (28, 38, 43, 44). Therefore, to illustrate the translational potential of brain-penetrating nanoparticles, we conducted a screen of ∼2,000 compounds that were previously used in Food and Drug Administration (FDA)-approved products for their ability to inhibit patient-derived BCSCs, encapsulated the best agents to emerge from the screen into brain-penetrating PLGA nanoparticles, and administered these nanocarriers by CED in a BCSC-derived xenograft model of GBM.     

Numb-deficient satellite cells have regeneration and proliferation defects

The adaptor protein Numb has been implicated in the switch between cell proliferation and differentiation made by satellite cells during muscle repair. Using two genetic approaches to ablate Numb, we determined that, in its absence, muscle regeneration in response to injury was impaired. Single myofiber cultures demonstrated a lack of satellite cell proliferation in the absence of Numb, and the proliferation defect was confirmed in satellite cell cultures. Quantitative RT-PCR from Numb-deficient satellite cells demonstrated highly up-regulated expression of p21 and Myostatin, both inhibitors of myoblast proliferation. Transfection with Myostatin-specific siRNA rescued the proliferation defect of Numb-deficient satellite cells. Furthermore, overexpression of Numb in satellite cells inhibited Myostatin expression. These data indicate a unique function for Numb during the initial activation and proliferation of satellite cells in response to muscle injury.Satellite cells represent a muscle-specific stem cell population that allows for muscle growth postnatally and is necessary for muscle repair (1). In response to muscle-fiber damage, quiescent satellite cells that lie along the myofibers under the plasmalemma are activated and proliferate. Proliferating satellite cells have a binary fate decision to make—they can differentiate into myoblasts and intercalate into myofibers by fusion to repair the damaged muscle or they can renew the satellite cell population and return to a quiescent state (2–4). Quiescent satellite cells express paired box 7 (Pax7), but low or undetectable levels of the myogenic regulatory factors Myf5 and MyoD (5, 6). Activated satellite cells robustly express Pax7 and MyoD/Myf5, but a subset will subsequently down-regulate the myogenic regulatory factors in the process of satellite cell self-renewal (7). Recent studies have demonstrated that, in vivo, Pax7-positive cells are necessary for muscle repair (8, 9).Notch signaling is an important regulator of satellite cell function; it is implicated in satellite cell activation, proliferation (2, 10, 11), and maintenance of quiescence (12, 13). Expression of constitutively active Notch1 results in maintenance of Pax7 expression and down-regulation of Myod/Myf5 whereas inhibition of Notch signaling leads to myogenic differentiation (10, 14). In fact, conditional ablation of Rbpj embryonically results in hypotrophic muscle (15), and, if ablated in the adult, satellite cells undergo spontaneous activation and precocious differentiation with a failure of self-renewal (12, 13). In adult muscle, the Notch ligand, Delta-like1 (Dll1), is expressed on satellite cells, myofibers, and newly differentiating myoblasts and is necessary for repair (10, 11, 16). In aged muscle, impairment of regeneration is due, in part, to a failure of Dll1 expression (17).Numb en`s four proteins with molecular masses of 65, 66, 71, and 72 kDa by alternative splicing of two exons (18, 19). The Numb proteins are cytoplasmic adaptors that direct ubiquitination and degradation of Notch1 by recruiting the E3 ubiquitin ligase Itch to the receptor (18–22). Numb is a cell-fate determinant that mediates asymmetric cell division, leading to selective Notch inhibition in one daughter cell and its subsequent differentiation whereas the other daughter has active Notch signaling and remains proliferative (10). Embryonically, Numb is expressed in the myotome whereas Notch1 is limited to the dermomyotome (23, 24). This pattern suggests that the expression of Numb in one daughter cell allows entry into the myogenic lineage. Indeed, overexpression of Numb embryonically increases the number of myogenic progenitors in the somite (25, 26).Numb expression increases during the activation and proliferative expansion of satellite cells, becoming asymmetrically segregated in transit-amplifying cells and leading to asymmetric cell divisions (10, 27). These observations led to a model in which Numb inhibits Notch signaling in one daughter satellite cell, allowing it to undergo myogenic differentiation. The molecular switch that controls the decision of satellite cell progeny to continue proliferating or to differentiate is not well understood. This process seems to be controlled by a decrease of Notch signaling due to increased expression of Numb and an increase in Wnt signaling (10–14, 17, 28). In these studies, we examined the role of Numb in satellite cell function by genetic deletion of Numb from myogenic progenitors and satellite cells. Our observations reveal that Numb is necessary for satellite cell-mediated repair. Furthermore, Numb-deficient satellite cells have an unexpected proliferation defect due to an up-regulation of Myostatin. These data indicate a unique role for Numb in regulating the activation and proliferation of satellite cells.     

Targeting RPL39 and MLF2 reduces tumor initiation and metastasis in breast cancer by inhibiting nitric oxide synthase signaling

We previously described a gene signature for breast cancer stem cells (BCSCs) derived from patient biopsies. Selective shRNA knockdown identified ribosomal protein L39 (RPL39) and myeloid leukemia factor 2 (MLF2) as the top candidates that affect BCSC self-renewal. Knockdown of RPL39 and MLF2 by specific siRNA nanoparticles in patient-derived and human cancer xenografts reduced tumor volume and lung metastases with a concomitant decrease in BCSCs. RNA deep sequencing identified damaging mutations in both genes. These mutations were confirmed in patient lung metastases (n = 53) and were statistically associated with shorter median time to pulmonary metastasis. Both genes affect the nitric oxide synthase pathway and are altered by hypoxia. These findings support that extensive tumor heterogeneity exists within primary cancers; distinct subpopulations associated with stem-like properties have increased metastatic potential.Large-scale sequencing analyses of solid cancers have identified extensive tumor heterogeneity within individual primary cancers (1). Recent studies indicate that such tumoral heterogeneity is associated with heterogeneous protein function, which fosters tumor adaptation, treatment resistance, and failure through Darwinian selection (2–4). Cancer stem cells are a subpopulation of cells within the primary tumor responsible for tumor initiation and metastases (5–9). Three groups have recently independently provided functional evidence for the presence of cancer stem cells by lineage-tracing experiments (10–12). These observations suggest that these subpopulations of cancer stem cells (CSCs) within the bulk primary tumor are resistant to conventional therapies through different adaptive mechanisms with the potential for self-renewal and metastases (7, 13, 14). However, few studies have determined the genetic profile of the cells that escape the primary cancer and evolve in distant metastatic sites (1). Additionally, no large-scale sequencing studies of metastases have been conducted because the majority of patients are treated with systemic therapies and not surgery.Tumor clonal heterogeneity within a primary tumor may in part be explained by hypoxic regions within the bulk tumor that have been correlated with invasiveness, therapeutic resistance, and metastasis (15–18). Cancer stem cells have been found to reside near hypoxic regions in some solid cancers (19–21). We have previously published a 477-gene tumorigenic signature by isolating breast cancer stem cells (BCSCs) derived from patient biopsies (22). Here, we have identified two previously unidentified cancer genes, ribosomal protein L39 (RPL39) and myeloid leukemia factor 2 (MLF2), by selective shRNA knockdown of genes from this tumorigenic signature, that impact breast cancer stem cell self-renewal and lung metastases. Analysis of 53 patient lung metastases confirmed damaging mutations in RPL39 and MLF2 in a significant number of samples, which conferred a gain-of-function phenotype. These mutations were statistically associated with shorter median time to distant relapse. We further describe a common mechanism of action through nitric oxide synthase signaling that is regulated by hypoxia.     

Toxin Kid uncouples DNA replication and cell division to enforce retention of plasmid R1 in Escherichia coli cells

Worldwide dissemination of antibiotic resistance in bacteria is facilitated by plasmids that encode postsegregational killing (PSK) systems. These produce a stable toxin (T) and a labile antitoxin (A) conditioning cell survival to plasmid maintenance, because only this ensures neutralization of toxicity. Shortage of antibiotic alternatives and the link of TA pairs to PSK have stimulated the opinion that premature toxin activation could be used to kill these recalcitrant organisms in the clinic. However, validation of TA pairs as therapeutic targets requires unambiguous understanding of their mode of action, consequences for cell viability, and function in plasmids. Conflicting with widespread notions concerning these issues, we had proposed that the TA pair kis-kid (killing suppressor-killing determinant) might function as a plasmid rescue system and not as a PSK system, but this remained to be validated. Here, we aimed to clarify unsettled mechanistic aspects of Kid activation, and of the effects of this for kis-kid–bearing plasmids and their host cells. We confirm that activation of Kid occurs in cells that are about to lose the toxin-encoding plasmid, and we show that this provokes highly selective restriction of protein outputs that inhibits cell division temporarily, avoiding plasmid loss, and stimulates DNA replication, promoting plasmid rescue. Kis and Kid are conserved in plasmids encoding multiple antibiotic resistance genes, including extended spectrum β-lactamases, for which therapeutic options are scarce, and our findings advise against the activation of this TA pair to fight pathogens carrying these extrachromosomal DNAs.Plasmids serve as extrachromosomal DNA platforms for the reassortment, mobilization, and maintenance of antibiotic resistance genes in bacteria, enabling host cells to colonize environments flooded with antimicrobials and to take advantage of resources freed by the extinction of nonresistant competitors. Fueled by these selective forces and aided by their itinerant nature, plasmids disseminate resistance genes worldwide shortly after new antibiotics are developed, which is a major clinical concern (1–3). However, in antibiotic-free environments, such genes are dispensable. There, the cost that plasmid carriage imposes on cells constitutes a disadvantage in the face of competition from other cells and, because plasmids depend on their hosts to survive, also a threat to their own existence.Many plasmids keep low copy numbers (CNs) to minimize the problem above, because it reduces burdens to host cells. However, this also decreases their chances to fix in descendant cells, a new survival challenge (4). To counteract this, plasmids have evolved stability functions. Partition systems pull replicated plasmid copies to opposite poles in host cells, facilitating their inheritance by daughter cells (5). Plasmids also bear postsegregational killing (PSK) systems, which encode a stable toxin and a labile antitoxin (TA) pair that eliminates plasmid-free cells produced by occasional replication or partition failures. Regular production of the labile antitoxin protects plasmid-containing cells from the toxin. However, antitoxin replenishment is not possible in cells losing the plasmid, and this triggers their elimination (5).TA pairs are common in plasmids disseminating antibiotic resistance in bacterial pathogens worldwide (2, 6–10). The link of these systems to PSK and the exiguous list of alternatives in the pipeline have led some to propose that chemicals activating these TA pairs may constitute a powerful antibiotic approach against these organisms (5, 11–13). However, the appropriateness of these TA pairs as therapeutic targets requires unequivocal understanding of their function in plasmids. Although PSK systems encode TA pairs, not all TA pairs might function as PSK systems, as suggested by their abundance in bacterial chromosomes, where PSK seems unnecessary (14–16). Moreover, the observation that many plasmids bear several TA pairs (6–10) raises the intriguing question of why they would need more than one PSK system, particularly when they increase the metabolic burden that plasmids impose on host cells (17). Because PSK functions are not infallible, their gathering may provide a mechanism for reciprocal failure compensation, minimizing the number of cells that escape killing upon plasmid loss (5). Alternatively, some TA pairs may stabilize plasmids by mechanisms different from PSK, and their grouping might not necessarily reflect functional redundancy (18).This may be the case in plasmid R1, which encodes TA pairs hok-sok (host killing-suppressor of killing) and kis(pemI)-kid(pemK) (19–23). Inconsistent with PSK, we had noticed that activation of toxin Kid occurred in cells that still contained R1, and that this happened when CNs were insufficient to ensure plasmid transmission to descendant cells. We also found that Kid cleaved mRNA at UUACU sites, which appeared well suited to trigger a response that prevented plasmid loss and increased R1 CNs without killing cells, as suggested by our results. In view of all this, we argued that Kid and Kis functioned as a rescue system for plasmid R1, and not as a PSK system (24). This proposal cannot be supported by results elsewhere, suggesting that Kid may cleave mRNA at simpler UAH sites (with H being A, C, or U) (25, 26), a view that has prevailed in the literature (14, 16, 27–29). Moreover, other observations indicate that our past experiments may have been inappropriate to conclude that Kid does not kill Escherichia coli cells (30, 31). Importantly, Kid, Kis, and other elements that we found essential for R1 rescue are conserved in plasmids conferring resistance to extended-spectrum β-lactamases, a worrying threat to human health (1, 6–10, 32). Therapeutic options to fight pathogens carrying these plasmids are limited, and activation of Kid may be perceived as a good antibiotic alternative. Because the potential involvement of this toxin in plasmid rescue advises against such approach, we aimed to ascertain here the mode of action; the effects on cells; and, ultimately, the function of Kid (and Kis) in R1.     

Noninvasive positron emission tomography and fluorescence imaging of CD133+ tumor stem cells

A technology that visualizes tumor stem cells with clinically relevant tracers could have a broad impact on cancer diagnosis and treatment. The AC133 epitope of CD133 currently is one of the best-characterized tumor stem cell markers for many intra- and extracranial tumor entities. Here we demonstrate the successful noninvasive detection of AC133⁺ tumor stem cells by PET and near-infrared fluorescence molecular tomography in subcutaneous and orthotopic glioma xenografts using antibody-based tracers. Particularly, microPET with ⁶⁴Cu-NOTA-AC133 mAb yielded high-quality images with outstanding tumor-to-background contrast, clearly delineating subcutaneous tumor stem cell-derived xenografts from surrounding tissues. Intracerebral tumors as small as 2–3 mm also were clearly discernible, and the microPET images reflected the invasive growth pattern of orthotopic cancer stem cell-derived tumors with low density of AC133⁺ cells. These data provide a basis for further preclinical and clinical use of the developed tracers for high-sensitivity and high-resolution monitoring of AC133⁺ tumor stem cells.Cancer stem cells (CSCs) are highly undifferentiated tumor cells with characteristics similar to normal stem cells. These characteristics include long-term replication, self-renewal, and aberrant differentiation (1, 2). Based on these characteristics, it has been hypothesized that only CSCs are able to propagate tumors for long periods of time and to initiate relapses or metastases. Furthermore, CSCs are considered to be more resistant to conventional radio- and chemotherapy than more differentiated tumor cells (3–5). Hence, elimination of CSCs is challenging but necessary for successful tumor eradication. The stem cell hypothesis of cancer development and progression is conceptually attractive and is supported by many preclinical (1, 2, 5–7) and some clinical studies (4, 8). However, larger clinical trials investigating the role of CSCs in patients have been hampered by the lack of techniques to detect, localize, and quantify the presence of CSCs noninvasively. Specifically, successful noninvasive imaging of unmanipulated CSCs with clinically relevant imaging probes (e.g., antibodies or other ligands binding CSC-specific cell-surface proteins) has not yet been reported (9–11).AC133 is an N-glycosylation–dependent epitope of the second extracellular loop of CD133/prominin-1, a cholesterol-binding protein of unknown function that locates to plasma membrane protrusions (12–14). Postnatally, the CD133 protein is expressed by certain epithelial and nonepithelial cells, by stem and progenitor cells of various organs, and by CSCs of many different types of malignant tumors (15). With a few exceptions, recognition of the AC133 epitope by the AC133 mAb appears to be limited to cells harboring stem cell properties, and the AC133 epitope—but not necessarily the CD133 protein—is down-regulated upon differentiation, presumably because of changes in glycosylation (12, 13, 15).AC133⁺ tumor stem cells have been described for glioblastoma multiforme (the most common and most aggressive primary brain tumor in adults), various pediatric brain and central nervous system tumors (medulloblastoma, ependymoma, pineoblastoma, teratoid/rhabdoid tumors, and retinoblastoma), brain metastases, many different types of carcinomas including colon, pancreatic, lung, liver, and ovarian cancer, melanoma, sarcomas, and different types of leukemia. Although AC133⁻ tumor stem cells also exist (16–18), AC133⁺ cells found in these and other tumor types have been shown to be able to self-renew, to differentiate, and to recreate the original tumors when injected into immunocompromised mice (8, 17, 19–22). Both, stemness and highly agressive malignant tumors often are associated with hypoxia (23), and hypoxia can promote the expansion of CD133⁺ cells (24). Therefore the frequent expression of AC133 on CSCs may reflect, in part, their common localization in a hypoxic environment (25).We previously reported the successful noninvasive detection of the AC133 epitope by antibody-based near-infrared fluorescence molecular tomography (NIR FMT) in mice with s.c. xenografts of CD133-overexpressing tumor cells or traditional tumor cell lines naturally displaying AC133 (26). However, we did not investigate patient-derived CSCs with the above-mentioned stem cell characteristics in that study, and NIR fluorescence, although penetrating tissues more deeply (2–4 cm) than visible-light fluorescence, has limited importance for clinical whole-body imaging (27).We report here the successful noninvasive detection of tumor-associated AC133 by PET, using a radiolabeled AC133-specific mAb in mice xenografted with tumor cell lines overexpressing CD133 or with patient-derived AC133⁺ CSCs. PET is highly sensitive and is widely used for clinical whole-body diagnostic imaging. As a PET nuclide, we used ⁶⁴Cu (t_1/2 = 12.7 h), which allows long-term tracking for at least 48 h, to follow the tumoral accumulation of relatively large molecules such as antibodies, that exhibit relatively slow tumor penetration (28). We chose S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA, hereafter abbreviated as NOTA) as the ⁶⁴Cu chelator, because high labeling efficiencies and high in vivo stability have been reported for ⁶⁴Cu-NOTA-antibody conjugates (29). In addition, we report the successful noninvasive detection of AC133⁺ CSCs with fluorescently labeled AC133 mAb and NIR imaging, a modality that is important for whole-body small-animal imaging and for intraoperative and endoscopic imaging and imaging of superficial tumors in humans (27, 30). In addition to imaging s.c. growing tumors, we report the successful antibody-mediated imaging of orthotopic xenografts initiated from AC133⁺ glioblastoma stem cells in the brain of immunocompromised mice, emphasizing the feasibility of noninvasive antibody-mediated imaging of brain tumors.     

Resveratrol and aspirin eliminate tetraploid cells for anticancer chemoprevention

Tetraploidy constitutes a genomically metastable state that can lead to aneuploidy and genomic instability. Tetraploid cells are frequently found in preneoplastic lesions, including intestinal cancers arising due to the inactivation of the tumor suppressor adenomatous polyposis coli (APC). Using a phenotypic screen, we identified resveratrol as an agent that selectively reduces the fitness of tetraploid cells by slowing down their cell cycle progression and by stimulating the intrinsic pathway of apoptosis. Selective killing of tetraploid cells was observed for a series of additional agents that indirectly or directly stimulate AMP-activated protein kinase (AMPK) including salicylate, whose chemopreventive action has been established by epidemiological studies and clinical trials. Both resveratrol and salicylate reduced the formation of tetraploid or higher-order polyploid cells resulting from the culture of human colon carcinoma cell lines or primary mouse epithelial cells lacking tumor protein p53 (TP53, best known as p53) in the presence of antimitotic agents, as determined by cytofluorometric and videomicroscopic assays. Moreover, oral treatment with either resveratrol or aspirin, the prodrug of salicylate, repressed the accumulation of tetraploid intestinal epithelial cells in the Apc^Min/+ mouse model of colon cancer. Collectively, our results suggest that the chemopreventive action of resveratrol and aspirin involves the elimination of tetraploid cancer cell precursors.One of the initiating triggers of carcinogenesis is illicit tetraploidization, i.e., the formation of cells that encompass twice as many chromosomes as their normal, diploid counterparts (1–4). Such an augmentation in nuclear DNA content may originate from cell-to-cell fusion, endocycling, or endomitosis. Contrasting with some exceptions (such as hepatocytes, syncytiotrophoblasts, megakaryocytes, and myocytes), most cell types do not tolerate significant variations from the diploid status, meaning that tetraploid as well as higher-order polyploid cells usually activate programmed death pathways as soon as they are generated (5) or elicit immune responses resulting in their elimination (6).A supraphysiological frequency of tetraploid cells has been detected at early stages of multiple cancer cell types (including bronchial, esophageal, gastric, mammary, colorectal, ovarian, cervical, and prostate carcinomas), often correlating with the inactivation of the tumor suppressors retinoblastoma 1 (RB1) and tumor protein p53 (TP53, best known as p53) (7). The inactivation of p53 facilitates the tetraploidization of cell lines (8–10) and primary epithelial cells from the colon and the mammary gland (11–13). Similarly, inactivation of the adenomatous polyposis coli (APC) tumor suppressor gene (whose mutations initiate a majority of colorectal cancers) results in tetraploidization both in vitro and in vivo in mouse models (14, 15).Tetraploid cells can give rise to an aneuploid offspring through several mechanisms, namely the gradual gain or loss of chromosomes during subsequent rounds of bipolar (and aberrant) mitosis or, alternatively, the reduction of the chromosomal content during multipolar mitoses (16). Such multipolar mitoses, which result from the presence of extra centrosomes, provoke asymmetric cell divisions in which chromosomes are close-to-randomly distributed among three or more daughter cells (12, 17). Exceptionally, newly generated aneuploid cells are fitter than their tetraploid progenitors, thus progressively transforming into malignant cells (2–5, 18).Given the importance of tetraploidization for oncogenesis, it is tempting to develop strategies for the selective eradication of such cells. Tetraploid cells are intrinsically resistant against DNA damaging agents (9), yet are more susceptible to a variety of agents including inhibitors of checkpoint kinase 1 (19), Aurora kinase B (20), and mitotic kinesins (21, 22). Nonetheless, such agents can perturb normal mitoses and mitotic checkpoints, casting doubts on their potential utility as chemopreventive agents. Driven by this consideration, we developed a screen for the identification of selective killers of tetraploid cells. This screen led to the identification of resveratrol and other AMP-activated protein kinase (AMPK) activators, including salicylate as potent antitetraploids.     

Dopamine release from transplanted neural stem cells in Parkinsonian rat striatum in vivo

Embryonic stem cell-based therapies exhibit great potential for the treatment of Parkinson’s disease (PD) because they can significantly rescue PD-like behaviors. However, whether the transplanted cells themselves release dopamine in vivo remains elusive. We and others have recently induced human embryonic stem cells into primitive neural stem cells (pNSCs) that are self-renewable for massive/transplantable production and can efficiently differentiate into dopamine-like neurons (pNSC–DAn) in culture. Here, we showed that after the striatal transplantation of pNSC–DAn, (i) pNSC–DAn retained tyrosine hydroxylase expression and reduced PD-like asymmetric rotation; (ii) depolarization-evoked dopamine release and reuptake were significantly rescued in the striatum both in vitro (brain slices) and in vivo, as determined jointly by microdialysis-based HPLC and electrochemical carbon fiber electrodes; and (iii) the rescued dopamine was released directly from the grafted pNSC–DAn (and not from injured original cells). Thus, pNSC–DAn grafts release and reuptake dopamine in the striatum in vivo and alleviate PD symptoms in rats, providing proof-of-concept for human clinical translation.Parkinson’s disease (PD) is a chronic progressive neurodegenerative disorder characterized by the specific loss of dopaminergic neurons in the substantia nigra pars compacta and their projecting axons, resulting in loss of dopamine (DA) release in the striatum (1). During the last two decades, cell-replacement therapy has proven, at least experimentally, to be a potential treatment for PD patients (2–7) and in animal models (8–15). The basic principle of cell therapy is to restore the DA release by transplanting new DA-like cells. Until recently, obtaining enough transplantable cells was a major bottleneck in the practicability of cell therapy for PD. One possible source is embryonic stem cells (ESCs), which can develop infinitely into self-renewable pluripotent cells with the potential to generate any type of cell, including DA neurons (DAns) (16, 17).Recently, several groups including us have introduced rapid and efficient ways to generate primitive neural stem cells (pNSCs) from human ESCs using small-molecule inhibitors under chemically defined conditions (12, 18, 19). These cells are nonpolarized neuroepithelia and retain plasticity upon treatment with neuronal developmental morphogens. Importantly, pNSCs differentiate into DAns (pNSC–DAn) with high efficiency (∼65%) after patterning by sonic hedgehog (SHH) and fibroblast growth factor 8 (FGF8) in vitro, providing an immediate and renewable source of DAns for PD treatment. Importantly, the striatal transplantation of human ESC-derived DA-like neurons, including pNSC–DAn, are able to relieve the motor defects in a PD rat model (11–13, 15, 19–23). Before attempting clinical translation of pNSC–DAn, however, there are two fundamental open questions. (i) Can pNSC–DAn functionally restore the striatal DA levels in vivo? (ii) What cells release the restored DA, pNSC–DAn themselves or resident neurons/cells repaired by the transplants?Regarding question 1, a recent study using nafion-coated carbon fiber electrodes (CFEs) reported that the amperometric current is rescued in vivo by ESC (pNSC–DAn-like) therapy (19). Both norepinephrine (NE) and serotonin are present in the striatum (24, 25). However, CFE amperometry/chronoamperometry alone cannot distinguish DA from other monoamines in vivo, such as NE and serotonin (Fig. S1) (see also refs. 26–28). Considering that the compounds released from grafted ESC-derived cells are unknown, the work of Kirkeby et al. was unable to determine whether DA or other monoamines are responsible for the restored amperometric signal. Thus, the key question of whether pNSC–DAn can rescue DA release needs to be reexamined for the identity of the restored amperometric signal in vivo.Regarding question 2, many studies have proposed that DA is probably released from the grafted cells (8, 12, 13, 20), whereas others have proposed that the grafted stem cells might restore striatal DA levels by rescuing injured original cells (29, 30). Thus, whether the grafted cells are actually capable of synthesizing and releasing DA in vivo must be investigated to determine the future cellular targets (residual cells versus pNSC–DAn) of treatment.To address these two mechanistic questions, advanced in vivo methods of DA identification and DA recording at high spatiotemporal resolution are required. Currently, microdialysis-based HPLC (HPLC) (31–33) and CFE amperometric recordings (34, 35) have been used independently by different laboratories to assess evoked DA release from the striatum in vivo. The major advantage of microdialysis-based HPLC is to identify the substances secreted in the cell-grafted striatum (33), but its spatiotemporal resolution is too low to distinguish the DA release site (residual cells or pNSC–DAn). In contrast, the major advantage of CFE-based amperometry is its very high temporal (ms) and spatial (μm) resolution, making it possible to distinguish the DA release site (residual cells or pNSC–DAn) in cultured cells, brain slices, and in vivo (34–39), but it is unable to distinguish between low-level endogenous oxidizable substances (DA versus serotonin and NE) in vivo.In the present study, we developed a challenging experimental paradigm of combining the two in vivo methods, microdialysis-based HPLC and CFE amperometry, to identify the evoked substance as DA and its release site as pNSC–DAn in the striatum of PD rats.