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. STEM CELLS 2008;26:1575-1586 Disclosure of potential conflicts of interest is found at the end of this article.

BackgroundTreatment 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. Iron-labeling 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.MethodologyFor 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 iron-loading 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.ConclusionFE-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”.

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.

ABSTRACT

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 starvationinduced suppression of c-jun NH 2 -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)

BackgroundRecombinant 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 FindingsAs 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.ConclusionsTaken 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.

BackgroundMetastasis 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.ResultsHere 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.ConclusionsOur 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.

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.