Due to their stem-like characteristics and their resistance to existing chemo- and radiation therapies, there is a growing appreciation that cancer stem cells (CSCs) are the root cause behind cancer ...metastasis and recurrence. However, these cells represent a small subpopulation of cancer cells and are difficult to propagate in vitro. Glioblastoma is an extremely deadly form of brain cancer that is hypothesized to have a subpopulation of CSCs called glioblastoma stem cells (GSCs; also called brain tumor initiating cells, BTICs). We propose the use of selective Rho-kinase (ROCK) inhibitors, Y-27632 and fasudil, to promote GSC/BTIC-like cell survival and propagation in vitro. ROCK inhibitors have been implicated in suppressing apoptosis, and it was hypothesized that they would increase the number of GSC/BTIC-like cells grown in vitro and improve cloning efficiencies. Indeed, our data demonstrate that transient and continuous supplementation of non-toxic concentrations of Y-27632 and fasudil inhibited apoptosis, enhanced the cells' ability to form spheres, and increased stem cell marker expressing GSC/BTIC-like cell subpopulation. Our data indicated that pharmacological and genetic (siRNA) inhibitions of the ROCK pathway facilitates in vitro expansion of GSC/BTIC-like cells. Thus, ROCK pathway inhibition shows promise for future optimization of CSC culture media.
Since the discovery of tumour initiating cells (TICs) in solid tumours, studies focussing on their role in cancer initiation and progression have abounded. The biological interrogation of these cells ...continues to yield volumes of information on their pro-tumourigenic behaviour, but actionable generalised conclusions have been scarce. Further, new information suggesting a dependence of tumour composition and growth on the microenvironment has yet to be studied theoretically. To address this point, we created a hybrid, discrete/continuous computational cellular automaton model of a generalised stem-cell driven tissue with a simple microenvironment. Using the model we explored the phenotypic traits inherent to the tumour initiating cells and the effect of the microenvironment on tissue growth. We identify the regions in phenotype parameter space where TICs are able to cause a disruption in homeostasis, leading to tissue overgrowth and tumour maintenance. As our parameters and model are non-specific, they could apply to any tissue TIC and do not assume specific genetic mutations. Targeting these phenotypic traits could represent a generalizable therapeutic strategy across cancer types. Further, we find that the microenvironmental variable does not strongly affect the outcomes, suggesting a need for direct feedback from the microenvironment onto stem-cell behaviour in future modelling endeavours.
Glioblastomas are the most prevalent and lethal primary brain tumor and are comprised of hierarchies with self-renewing cancer stem cells (CSCs) at the apex. Like neural stem cells (NSCs), CSCs ...reside in functional niches that provide essential cues to maintain the cellular hierarchy. Bone morphogenetic proteins (BMPs) instruct NSCs to adopt an astrocyte fate and are proposed as anti-CSC therapies to induce differentiation, but, paradoxically, tumors express high levels of BMPs. Here we demonstrate that the BMP antagonist Gremlinl is specifically expressed by CSCs as protection from endogenous BMPs. Gremlinl colocalizes with CSCs in vitro and in vivo. Furthermore, Gremlinl blocks prodifferentiation effects of BMPs, and overexpression of Gremlinl in non-CSCs decreases their endogenous BMP signaling to promote stem-like features. Consequently, Gremlin 1-overexpressing cells display increased growth and tumor formation abilities. Targeting Gremlinl in CSCs results in impaired growth and self-renewal. Transcriptional profiling demonstrated that Gremlinl effects were associated with inhibition of p2l super(WAF1/>CIP1); a key CSC signaling node. This study establishes CSC-derived Gremlinl as a driving force in maintaining glioblastoma tumor proliferation and glioblastoma hierarchies through the modulation of endogenous prodifferentiation signals.
Abstract
Growth factor-mediated proliferation and self-renewal maintains tissue specific stem cells and is frequently dysregulated in cancers. Platelet-derived growth factor ligands and receptors ...(PDGFRs) are commonly overexpressed in gliomas and initiate tumors in genetically engineered models. PDGFRα alterations inform intertumoral heterogeneity towards a proneural glioma subgroup. We interrogated the role of PDGFRs in intratumoral heterogeneity with regards to differences in tumor cells. We found that PDGFRα is expressed only in a subset of gliomas while PDGFRβ is more commonly expressed in tumors but largely restricted to self-renewing tumorigenic glioma stem cells (GSCs). Targeting PDGFRβ genetically or with specific inhibitors attenuated GSC self-renewal, survival, tumor growth, and invasion. PDGFRβ targeting was associated with decreased activation of the cancer stem cell signaling node STAT3, and STAT3 activity rescued loss of PDGFRβ expression. These results demonstrate that a growth factor receptor family can differentially function to promote tumor heterogeneity. Our results may explain mixed clinical responses of anti-PDGFR based approaches, and suggest the need for integration of models of cancer as an organ system into development of cancer therapies.
Citation Format: {Authors}. {Abstract title} abstract. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5192. doi:1538-7445.AM2012-5192
Nitric oxide (NO) contributes to a wide variety of intracellular signaling and cell stress pathways in mammalian cells. Experimental manipulation of NO is frequently achieved by inhibition or ...activation of the NO synthase (NOS) family of enzymes responsible for intracellular NO production, or through employment of relatively nonspecific NO‐reactive compounds or antioxidants. Here we introduce a novel methodology to manipulate NO levels in mammalian systems via heterologous expression of the prokaryotic NO‐consuming flavohemoglobin (FlavoHb). This enzyme is an NO‐specific dioxygenase that converts NO to inert nitrate under aerobic conditions, thus functioning to consume NO and protect microbes from nitrosative stress. We demonstrate that FlavoHb can be employed in a similar fashion in mammalian cells to inhibit both NO‐dependent signal transduction and nitrosative stress. This strategy provides a highly specific and novel method for modulating intracellular NO levels and represents an alternative or complimentary strategy to utilization of NOS inhibitors or promiscuous antioxidants. Importantly, FlavoHb expression is well tolerated by mammalian cells with no evidence of NO‐independent toxicity. Future studies of NO in mammalian systems could employ FlavoHb as either a primary experimental or a confirmatory tool for probing functional and molecular consequences of NO within cells.
Glioblastomas are deadly cancers that display a functional cellular hierarchy maintained by self-renewing glioblastoma stem cells (GSCs). GSCs are regulated by molecular pathways distinct from the ...bulk tumor that may be useful therapeutic targets. We determined that A20 (TNFAIP3), a regulator of cell survival and the NF-κB pathway, is overexpressed in GSCs relative to non-stem glioblastoma cells at both the mRNA and protein levels. To determine the functional significance of A20 in GSCs, we targeted A20 expression with lentiviral-mediated delivery of short hairpin RNA (shRNA). Inhibiting A20 expression decreased GSC growth and survival through mechanisms associated with decreased cell-cycle progression and decreased phosphorylation of p65/RelA. Elevated levels of A20 in GSCs contributed to apoptotic resistance: GSCs were less susceptible to TNFα-induced cell death than matched non-stem glioma cells, but A20 knockdown sensitized GSCs to TNFα-mediated apoptosis. The decreased survival of GSCs upon A20 knockdown contributed to the reduced ability of these cells to self-renew in primary and secondary neurosphere formation assays. The tumorigenic potential of GSCs was decreased with A20 targeting, resulting in increased survival of mice bearing human glioma xenografts. In silico analysis of a glioma patient genomic database indicates that A20 overexpression and amplification is inversely correlated with survival. Together these data indicate that A20 contributes to glioma maintenance through effects on the glioma stem cell subpopulation. Although inactivating mutations in A20 in lymphoma suggest A20 can act as a tumor suppressor, similar point mutations have not been identified through glioma genomic sequencing: in fact, our data suggest A20 may function as a tumor enhancer in glioma through promotion of GSC survival. A20 anticancer therapies should therefore be viewed with caution as effects will likely differ depending on the tumor type.
Abstract
Although current therapeutic strategies for malignant gliomas are non-specifically directed against the tumor bulk, increasing evidence suggests that a single glioma contains a range of ...phenotypically disparate cell populations with differential capacity for tumor maintenance and initiation. Recent work in brain tumors suggests the existence of cellular sub-populations (known as cancer stem cells, or CSCs) that possess stem cell-like behaviors and a potent capacity for tumor initiation in transplantation assays relative to non-CSCs. Specifically targeting the highly tumorigenic CSCs has the potential to improve the prognosis of malignant glioma patients, but many molecularly-directed anti-CSC agents may also compromise the survival of normal neural stem cells due to molecular similarities between CSCs and normal stem cells. Here we demonstrate that malignant glioma CSCs express high levels of a particular protein relative to non-CSCs. Glioma CSC growth and tumorigenic capacity were abrogated by multiple strategies interfering with the expression and activity of this protein, including RNA interference, pharmacological inhibition, and heterologous expression of a prokaryotic enzyme that specifically consumes the enzymatic product of this critical protein. Importantly, tumor growth was inhibited by systemic treatment of xenograft-bearing mice with a well-studied small molecule inhibitor of this protein that has already demonstrated low toxicity in clinical trials for other diseases. Though the activity of this protein appears critical for the maintenance of highly tumorigenic glioma CSCs, our examination of both normal human neural progenitor cells and neural stem cells derived from genetically disrupted mice indicate no substantial role for this protein in normal neural stem cells. Our findings identify a novel glioma therapeutic target with a negligible role in normal neural stem cells, suggesting clinical utility for selective inhibitors of this CSC-specific molecular target.
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 3318.
Glioblastomas display cellular hierarchies containing tumor-propagating glioblastoma stem cells (GSCs). STAT3 is a critical signaling node in GSC maintenance but molecular mechanisms underlying STAT3 ...activation in GSCs are poorly defined. Here we demonstrate that the non-receptor tyrosine kinase BMX activates STAT3 signaling to maintain self-renewal and tumorigenic potential of GSCs. BMX is differentially expressed in GSCs relative to non-stem cancer cells and neural progenitors. BMX knockdown potently inhibited STAT3 activation, expression of GSC transcription factors, and growth of GSC-derived intracranial tumors. Constitutively active STAT3 rescued the effects of BMX downregulation, supporting that BMX signals through STAT3 in GSCs. These data demonstrate that BMX represents a GSC therapeutic target and reinforces the importance of STAT3 signaling in stem-like cancer phenotypes.
Runx 2 (runt-related transcription factor 2) is a master regulator of skeletogenesis. Distinct promoters in the Runx2 gene transcribe
the âbone-relatedâ Runx 2-II and non-osseous Runx 2-I ...isoforms that differ only in their respective N termini. Existing mutant mouse models with both isoforms deleted exhibit
an arrest of osteoblast and chondrocyte maturation and the complete absence of mineralized bone, but they do not distinguish
the separate functions of the two N-terminal isoforms. To elucidate the function of the bone-related isoform, we generated
selective Runx 2-II-deficient mice by the targeted deletion of the distal promoter and exon 1. Homozygous Runx 2-II-deficient ( Runx 2-II -/- ) mice unexpectedly formed axial, appendicular, and craniofacial bones derived from either intramembranous ossification or
mesenchymal cells of the bone collar, but they failed to form the posterior cranium and other bones derived from endochondral
ossification. Heterozygous Runx 2-II-deficient mice had grossly normal skeletons, but were osteopenic. The commitment of mesenchymal cells ex vivo to the osteoblast lineage occurred in Runx 2-II -/- mice, but osteoblastic gene expression was impaired. Chondrocyte maturation appeared normal, but the zone of hypertrophic
chondrocytes was not transformed into metaphyseal bone, leading to widened growth plates in Runx 2-II -/- mice. Compensatory increments in Runx 2-I expression occurred in Runx 2-II -/- mice but were not sufficient to normalize osteoblastic maturation or transcriptional activity. Our findings support distinct
functions of Runx 2-II and -I in the control of skeletogenesis. Runx 2-I is sufficient for early osteoblastogenesis and intramembranous bone formation, whereas Runx 2-II is necessary for complete osteoblastic maturation and endochondral bone formation.
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