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Supplementary Figures 1 and 2 from Bone Morphogenetic Protein 7 Protects Prostate Cancer Cells from Stress-Induced Apoptosis via Both Smad and c-Jun NH2-Terminal Kinase Pathways

We reported earlier that exposure to exogenous bone morphogenetic protein 7 (BMP7) could strongly inhibit serum starvation–induced apoptosis to C4-2B cell line, a variant of the LNCaP human prostate cancer cell line with propensity for bone metastasis. Whereas serum starvation suppressed the expression of survivin, a member of the inhibitor of apoptosis protein family, its expression was sustained in the presence of BMP7. In this study, we present evidence that BMP7 exposure up-regulated survivin promoter activity, an effect that was associated with activation of Smad, and could be repressed by dominant-negative Smad5. Additionally, serum starvation–induced suppression of c-jun NH2-terminal kinase (JNK) activity in C4-2B cells could be mostly restored by BMP7, and a JNK inhibitor could counteract the antiapoptotic effect of BMP7, without a significant effect on the level of survivin expression. Thus, we identified JNK pathway as another signaling mode for the antiapoptotic function of BMP7. To test the effect of endogenous up-regulation of BMP7, we genetically modulated the C4-2B cell line to overexpress BMP7 protein. Not only was this altered cell line resistant to serum starvation–induced apoptosis but it also exhibited patterns of Smad activation, survivin up-regulation, and JNK activation similar to those of the parental C4-2B cells exposed to exogenous BMP7. Consistent with these in vitro findings of BMP7 action, we acquired correlative results of Smad activation, survivin expression, and JNK activation in the progression of prostate cancer in the conditional Pten deletion mouse model, in which we first obtained the evidence of BMP7 overexpression. (Cancer Res 2006; 66(8): 4285-90)

Human neural and mesenchymal stem cells have been identified for cell-based therapies in regenerative medicine and as vehicles for delivering therapeutic agents to areas of injury and tumors. However, the signals required for homing and recruitment of stem cells to these sites are not well understood. Urokinase plasminogen activator (uPA) and urokinase plasminogen activator receptor (uPAR) are involved in chemotaxis and cell guidance during normal development and are upregulated in invasive tumors. Here we provided evidence that activation of uPA and uPAR in malignant solid tumors (brain, lung, prostate, and breast) augments neural and mesenchymal stem cell tropism. Expression levels of uPAR on human solid tumor cell lines correlated with levels of uPA and soluble uPAR in tumor cell-conditioned media. Cytokine expression profiles of these tumor-conditioned media were determined by protein arrays. Among 79 cytokines investigated, interleukin (IL)-6, IL-8, and monocyte chemoattractant protein-1 were the most highly expressed cytokines in uPAR-positive tumors. We provided evidence that human recombinant uPA induced stem cell migration, whereas depletion of uPA from PC-3 prostate cancer cell-conditioned medium blocked stem cell migration. Furthermore, retrovirus-mediated overexpression of uPA and uPAR in neuroblastoma (NB1691) cells induced robust migration of stem cells toward NB1691 cell-conditioned media, compared with media derived from wild-type NB1691 cells. We conclude that expression of uPA and uPAR in cancer cells underlies a novel mechanism of stem cell tropism to malignant solid tumors, which may be important for development of optimal stem cell-based therapies. Disclosure of potential conflicts of interest is found at the end of this article.

The utility of neural stem cells (NSCs) has extended beyond regenerative medicine to targeted gene delivery, as NSCs possess an inherent tropism to solid tumors, including invasive gliomas. However, for optimal clinical implementation, an understanding of the molecular events that regulate NSC tumor tropism is needed to ensure their safety and to maximize therapeutic efficacy. We show that human NSC lines responded to multiple tumor-derived growth factors and that hepatocyte growth factor (HGF) induced the strongest chemotactic response. Gliomatropism was critically dependent on c-Met signaling, as short hairpin RNA-mediated ablation of c-Met significantly attenuated the response. Furthermore, inhibition of Ras-phosphoinositide 3-kinase (PI3K) signaling impaired the migration of human neural stem cells (hNSCs) toward HGF and other growth factors. Migration toward tumor cells is a highly regulated process, in which multiple growth factor signals converge on Ras-PI3K, causing direct modification of the cytoskeleton. The signaling pathways that regulate hNSC migration are similar to those that promote unregulated glioma invasion, suggesting shared cellular mechanisms and responses. Disclosure of potential conflicts of interest is found at the end of this article.

We reported earlier that exposure to exogenous bone morphogenetic protein 7 (BMP7) could strongly inhibit serum starvation–induced apoptosis to C4-2B cell line, a variant of the LNCaP human prostate cancer cell line with propensity for bone metastasis. Whereas serum starvation suppressed the expression of survivin, a member of the inhibitor of apoptosis protein family, its expression was sustained in the presence of BMP7. In this study, we present evidence that BMP7 exposure up-regulated survivin promoter activity, an effect that was associated with activation of Smad, and could be repressed by dominant-negative Smad5. Additionally, serum starvation–induced suppression of c-jun NH2-terminal kinase (JNK) activity in C4-2B cells could be mostly restored by BMP7, and a JNK inhibitor could counteract the antiapoptotic effect of BMP7, without a significant effect on the level of survivin expression. Thus, we identified JNK pathway as another signaling mode for the antiapoptotic function of BMP7. To test the effect of endogenous up-regulation of BMP7, we genetically modulated the C4-2B cell line to overexpress BMP7 protein. Not only was this altered cell line resistant to serum starvation–induced apoptosis but it also exhibited patterns of Smad activation, survivin up-regulation, and JNK activation similar to those of the parental C4-2B cells exposed to exogenous BMP7. Consistent with these in vitro findings of BMP7 action, we acquired correlative results of Smad activation, survivin expression, and JNK activation in the progression of prostate cancer in the conditional Pten deletion mouse model, in which we first obtained the evidence of BMP7 overexpression. (Cancer Res 2006; 66(8): 4285-90)

Reports of non-neural differentiation of neural stem cells (NSCs) have been challenged by alternative explanations for expanded differentiation potentials. In an attempt to demonstrate the plasticity of NSC, neurospheres were generated from single retrovirally labeled embryonic cortical precursors. In a defined serum-free insulin-containing media, 40% of the neurospheres contained both myogenic and neurogenic differentiated progeny. The number of NSCs displaying multilineage differentiation potential declines through gestation but does exist in the adult animal. In this system, insulin appears to function as a survival and dose-dependent myogenic differentiation signal for multilineage NSCs (MLNSC). MLNSC-derived cardiomyocytes contract synchronously, respond to sympathetic and parasympathetic stimulation, and regenerate injured heart tissues. These studies provide support for the hypothesis that MLNSCs exist throughout the lifetime of the animal, and potentially provide a population of stem cells for cell-based regenerative medicine strategies inside and outside of the nervous system.

Background: Glioblastoma multiforme (GBM) is the most aggressive type of malignant primary brain tumors in adults. Molecular and genetic analysis has advanced our understanding of glioma biology, however mapping the cellular composition of the tumor microenvironment is crucial for understanding the pathology of this dreaded brain cancer. In this study we identified major cell populations attracted by glioma using orthotopic rodent models of human glioma xenografts. Marker-specific, anatomical and morphological analyses revealed a robust influx of host cells into the main tumor bed and tumor satellites. Methodology/Principal Findings: Human glioma cell lines and glioma spheroid orthotopic implants were used in rodents. In both models, the xenografts recruited large numbers of host nestin-expressing cells, which formed a ‘network’ with glioma. The host nestin-expressing cells appeared to originate in the subventricular zone ipsilateral to the tumor, and were clearly distinguishable from pericytes that expressed smooth muscle actin. These distinct cell populations established close physical contact in a ‘pair-wise’ manner and migrated together to the deeper layers of tumor satellites and gave rise to tumor vasculature. The GBM biopsy xenografts displayed two different phenotypes: (a) low-generation tumors (first in vivo passage in rats) were highly invasive and non-angiogenic, and host nestin-positive cells that infiltrated into these tumors displayed astrocytic or elongated bipolar morphology; (b) high-generation xenografts (fifth passage) had pronounced cellularity, were angiogenic with ‘glomerulus-like’ microvascular proliferations that contained host nestin-positive cells. Stromal cell-derived factor-1 and its receptor CXCR4 were highly expressed in and around glioma xenografts, suggesting their role in glioma progression and invasion. Conclusions/Significance: Our data demonstrate a robust migration of nestin-expressing host cells to glioma, which together with pericytes give rise to tumor vasculature. Mapping the cellular composition of glioma microenvironment and deciphering the complex ‘crosstalk’ between tumor and host may ultimately aid the development of novel anti-glioma therapies. publishedVersion

Multiple myeloma (MM) is an incurable plasma cell malignancy where nearly all patients succumb to a relapse. The current preclinical models of MM target the plasma cells, constituting the bulk of the tumor, leaving the cancer stem cells to trigger a relapse. Utilizing a three-dimensional tissue culture system where cells were grown in extracellular matrix designed to reconstruct human bone marrow, we tested the anti-multiple myeloma cancer stem cell (MM-CSC) potential of two natural product inhibitors of nuclear factor κB (NFκB). Here we show that parthenolide and andrographolide are potent anti-MM-CSC agents. Both natural products demonstrated preferential toxicity toward MM-CSCs over non-tumorigenic MM cells. Addition of the bone marrow stromal compartment abrogated andrographolide activity while having no effect on parthenolide cytoxicity. This is the first report of a natural product with anti-CSC activity in myeloma, suggesting that it has the potential to improve the survival of patients with MM by eliminating the relapse-causing MM-CSCs.

Background: Treatment strategies for the highly invasive brain tumor, glioblastoma multiforme, require that cells which have invaded into the surrounding brain be specifically targeted. The inherent tumor-tropism of neural stem cells (NSCs) to primary and invasive tumor foci can be exploited to deliver therapeutics to invasive brain tumor cells in humans. Use of the strategy of converting prodrug to drug via therapeutic transgenes delivered by immortalized therapeutic NSC lines have shown efficacy in animal models. Thus therapeutic NSCs are being proposed for use in human brain tumor clinical trials. In the context of NSC-based therapies, MRI can be used both to non-invasively follow dynamic spatio-temporal patterns of the NSC tumor targeting allowing for the optimization of treatment strategies and to assess efficacy of the therapy. Ironlabeling of cells allows their presence to be visualized and tracked by MRI. Thus we aimed to iron-label therapeutic NSCs without affecting their cellular physiology using a method likely to gain United States Federal Drug Administration (FDA) approval. Methodology: For human use, the characteristics of therapeutic Neural Stem Cells must be clearly defined with any pertubation to the cell including iron labeling requiring reanalysis of cellular physiology. Here, we studied the effect of ironloading of the therapeutic NSCs, with ferumoxide-protamine sulfate complex (FE-Pro) on viability, proliferation, migratory properties and transgene expression, when compared to non-labeled cells. FE-Pro labeled NSCs were imaged by MRI at tumor sites, after intracranial administration into the hemisphere contralateral to the tumor, in an orthotopic human glioma xenograft mouse model. Conclusion: FE-Pro labeled NSCs retain their proliferative status, tumor tropism, and maintain stem cell character, while allowing in vivo cellular MRI tracking at 7 Tesla, to monitor their real-time migration and distribution at brain tumor sites. Of significance, this work directly supports the use of FE-Pro-labeled NSCs for real-time tracking in the clinical trial under development: ‘‘A Pilot Feasibility Study of Oral 5-Fluorocytosine and Genetically modified Neural Stem Cells Expressing Escherichia coli Cytosine Deaminase for Treatment of Recurrent High-Grade Gliomas’’.

Neural stem cells (NSCs) hold great promise for glioma therapy due to their inherent tumor-tropic properties, enabling them to deliver therapeutic agents directly to invasive tumor sites. In the present study, we visualized and quantitatively analyzed the spatial distribution of tumor-tropic NSCs in a mouse model of orthotopic glioma in order to predict the therapeutic efficacy of a representative NSC-based glioma therapy. U251.eGFP human glioma was established in the brain of athymic mice, followed by stereotactic injection of CM-DiI-labeled human NSCs posterior-lateral to the tumor site. Confocal microscopy, three-dimensional modeling and mathematical algorithms were used to visualize and characterize the spatial distribution of NSCs throughout the tumor. The pattern of NSC distribution showed a gradient with higher densities toward the centroid of the tumor mass. We estimate that NSC-mediated therapy would eradicate 70-90% of the primary tumor mass and the majority of invasive tumor foci. Our method may serve as a model for optimizing the efficacy of NSC-based glioma therapy.

Understanding the molecular events that govern neural progenitor lineage commitment, mitotic arrest, and differentiation into functional progeny are germane to our understanding of neocortical development. Members of the family of bone morphogenetic proteins (BMPs) play pivotal roles in regulating neural differentiation and apoptosis during neurogenesis through combined actions involving Smad and TAK1 activation. We demonstrate that BMP signaling is required for the induction of apoptosis of neural progenitors and that NRAGE is a mandatory component of the signaling cascade. NRAGE possesses the ability to bind and function with the TAK1-TAB1-XIAP complex facilitating the activation of p38. Disruption of NRAGE or any other member of the noncanonical signaling cascaded is sufficient to block p38 activation and thus the proapoptotic signals generated through BMP exposure. The function of NRAGE is independent of Smad signaling, but the introduction of a dominant-negative Smad5 also rescues neural progenitor apoptosis, suggesting that both canonical and noncanonical pathways can converge and regulate BMP-mediated apoptosis. Collectively, these results establish NRAGE as an integral component in BMP signaling and clarify its role during neural progenitor development.

The p75 neurotrophin receptor (p75NTR) is a member of the tumor necrosis factor receptor superfamily. p75NTR signaling events have been implicated in both cell cycle arrest and apoptosis depending on which effector molecules are associated with its intracellular domain after ligand binding. Two such effector proteins, p75NTR-associated cell death executor (NADE) and neurotrophin receptor interacting factor (NRIF) promote p75NTR-mediated apoptosis, whereas Schwann cell factor-1 (SC-1) mediates neurotrophin-dependent withdrawal from the cell cycle. An understanding of the expression profiles of these three interacting proteins and p75NTR during embryogenesis is critical for addressing whether these effector proteins might function outside of p75NTR-mediated signaling events. The distribution of NADE, NRIF and SC-1 mRNAs during murine development suggests that the action of these genes is in fact not limited to regions of p75NTR expression. Specifically, a detailed comparison of the spatial and temporal expression domains of NADE, NRIF and SC-1 during brain development revealed regions of co-expression with p75NTR but also illustrates a distinct and discordant spatial and temporal expression. These results yield novel insights into the unique developmental characteristics of the three p75NTR-interacting proteins, thus revealing their diverse signaling potential during embryonic development.

Neurotrophin receptor-interacting MAGE (NRAGE) is the most recently identified p75 neurotrophin receptor (p75NTR) intracellular binding protein. Previously, NRAGE over-expression was shown to mediate cell cycle arrest and facilitate nerve growth factor (NGF) dependent apoptosis of sympathetic neuroblasts in a p75NTR specific manner. Here we have examined the temporal and spatial expression patterns of NRAGE over the course of murine embryogenesis to determine whether NRAGE's expression is consistent with its proposed functions. We demonstrate that NRAGE mRNA and protein are expressed throughout embryonic and adult tissues. The mRNA is constitutively expressed within each tissue across development. However, expression of NRAGE protein displays a tight temporal tissue specific regulation. During early CNS development, NRAGE protein is expressed throughout the neural tube, but by later stages of neurogenesis, NRAGE protein is restricted within the ventricular zone, subplate and cortical plate. Moreover, NRAGE protein expression is limited to proliferative neural subpopulations as we fail to detect NRAGE expression co-localized with mature/differentiation associated neuronal markers. Interestingly, NRAGE's expression is not restricted solely to areas of p75NTR expression suggesting that NRAGE may mediate proliferation and/or apoptosis from other environmental signals in addition to NGF within the CNS. Our data support previously characterized roles for NRAGE as a mediator of precursor apoptosis and a repressor of cell cycle progression in neural development.

Background Recombinant monoclonal antibodies have emerged as important tools for cancer therapy. Despite the promise shown by antibody-based therapies, the large molecular size of antibodies limits their ability to efficiently penetrate solid tumors and precludes efficient crossing of the blood-brain-barrier into the central nervous system (CNS). Consequently, poorly vascularized solid tumors and CNS metastases cannot be effectively treated by intravenously-injected antibodies. The inherent tumor-tropic properties of human neural stem cells (NSCs) can potentially be harnessed to overcome these obstacles and significantly improve cancer immunotherapy. Intravenously-delivered NSCs preferentially migrate to primary and metastatic tumor sites within and outside the CNS. Therefore, we hypothesized that NSCs could serve as an ideal cellular delivery platform for targeting antibodies to malignant tumors. Methods and findings As proof-of-concept, we selected Herceptin (trastuzumab), a monoclonal antibody widely used to treat HER2-overexpressing breast cancer. HER2 overexpression in breast cancer is highly correlated with CNS metastases, which are inaccessible to trastuzumab therapy. Therefore, NSC-mediated delivery of trastuzumab may improve its therapeutic efficacy. Here we report, for the first time, that human NSCs can be genetically modified to secrete anti-HER2 immunoglobulin molecules. These NSC-secreted antibodies assemble properly, possess tumor cell-binding affinity and specificity, and can effectively inhibit the proliferation of HER2-overexpressing breast cancer cells in vitro. We also demonstrate that immunoglobulin-secreting NSCs exhibit preferential tropism to tumor cells in vivo, and can deliver antibodies to human breast cancer xenografts in mice. Conclusions Taken together, these results suggest that NSCs modified to secrete HER2-targeting antibodies constitute a promising novel platform for targeted cancer immunotherapy. Specifically, this NSC-mediated antibody delivery system has the potential to significantly improve clinical outcome for patients with HER2-overexpressing breast cancer.

Background Metastasis is the primary cause of death for cancer patients. TWIST1, an evolutionarily conserved basic helix-loop-helix (bHLH) transcription factor, is a strong promoter of metastatic spread and its expression is elevated in many advanced human carcinomas. However, the molecular events triggered by TWIST1 to motivate dissemination of cancer cells are largely unknown. Results Here we show that TWIST1 induces the production of interleukin 8 (IL8), which activates matrix metalloproteinases and promotes invasion of breast epithelial and cancer cells. In this novel mechanism, TWIST1-mediated IL8 transcription is induced through the TWIST1 carboxy-terminal WR (Trp-Arg) domain instead of the classic DNA binding bHLH domain. Co-immunoprecipitation analyses revealed that the WR domain mediates the formation of a protein complex comprised of TWIST1 and the nuclear factor-kappaB (NF-κB) subunit RELA (p65/NF-κB3), which synergistically activates the transcriptional activity of NF-κB. This activation leads to increased DNA binding affinity of RELA to the IL8 promoter and thus induces the expression of the cytokine. Blockage of IL8 signaling by IL8 neutralizing antibodies or receptor inhibition reduced the invasiveness of both breast epithelial and cancer cells, indicating that TWIST1 induces autonomous cell invasion by establishing an IL8 antocrine loop. Conclusions Our data demonstrate that the TWIST1 WR domain plays a critical role in TWIST1-induced IL8 expression through interactions with and activation of NF-κB. The produced IL8 signals through an autocrine loop and promotes extracellular matrix degradation to enable cell invasion across the basement membrane.