The Promise of MicroRNA Replacement Therapy BADER, Andreas G; BROWN, David; WINKLES, Matthew
Cancer research (Chicago, Ill.),
09/2010, Volume:
70, Issue:
18
Journal Article
Peer reviewed
Open access
MicroRNAs (miRNA), a class of natural RNA-interfering agents, have recently been identified as attractive targets for therapeutic intervention. The rationale for developing miRNA therapeutics is ...based on the premise that aberrantly expressed miRNAs play key roles in the development of human disease, and that correcting these miRNA deficiencies by either antagonizing or restoring miRNA function may provide a therapeutic benefit. Although miRNA antagonists are conceptually similar to other inhibitory therapies, restoring the function of a miRNA by miRNA replacement is a less well characterized approach. Here, we discuss the specific properties of miRNA replacement and review recent examples that explored the therapeutic delivery of miRNA mimics in animal models of cancer.
Fibroblast growth factor-inducible 14 (Fn14) is a highly inducible cytokine receptor that engages multiple intracellular signaling pathways, including nuclear factor-κB (NF-κB) and mitogen-activated ...protein kinase (MAPK). Fn14 expression is regulated by several cytokines and growth factors, and Fn14 is transiently up-regulated after injury. In contrast, in states of chronic inflammatory disease and in some solid tumors, Fn14 is persistently up-regulated. However, the post-translational regulation of Fn14 expression has not been directly investigated. Thus, we examined Fn14 proteostasis in the presence and absence of the Fn14 ligand TNF-like weak inducer of apoptosis (TWEAK). Similar to other TNF receptor superfamily members, we found that TWEAK induces Fn14 internalization and degradation. Surprisingly, we also observed rapid, TWEAK-independent, constitutive Fn14 internalization and turnover. Fn14 levels are maintained in cell culture by ongoing synthesis and trafficking of the receptor, leading to subsequent down-regulation by lysosomal degradation. Unexpectedly, the extracellular domain of Fn14 is necessary and sufficient for constitutive turnover. Based on these findings, we propose a model in which constitutive down-regulation of Fn14 facilitates dynamic regulation of Fn14 protein levels and prevents spontaneous or inappropriate receptor signaling.
Fn14 is a highly inducible TNF superfamily cytokine receptor.
Fn14 undergoes rapid, ligand-independent internalization and degradation mediated by the extracellular domain of the receptor.
Fn14 expression is regulated through transcription as described previously and through a novel post-translational mechanism.
Receptor trafficking may play an important role in regulating receptor availability, cytokine responses, and ligand-independent signaling.
Malignant glioblastomas are characterized by their ability to infiltrate into normal brain. We previously reported that binding of the multifunctional cytokine TNF-like weak inducer of apoptosis ...(TWEAK) to its receptor fibroblast growth factor-inducible 14 (Fn14) induces glioblastoma cell invasion via Rac1 activation. Here, we show that Cdc42 plays an essential role in Fn14-mediated activation of Rac1. TWEAK-treated glioma cells display an increased activation of Cdc42, and depletion of Cdc42 using siRNA abolishes TWEAK-induced Rac1 activation and abrogates glioma cell migration and invasion. In contrast, Rac1 depletion does not affect Cdc42 activation by Fn14, showing that Cdc42 mediates TWEAK-stimulated Rac1 activation. Furthermore, we identified two guanine nucleotide exchange factors (GEF), Ect2 and Trio, involved in TWEAK-induced activation of Cdc42 and Rac1, respectively. Depletion of Ect2 abrogates both TWEAK-induced Cdc42 and Rac1 activation, as well as subsequent TWEAK-Fn14-directed glioma cell migration and invasion. In contrast, Trio depletion inhibits TWEAK-induced Rac1 activation but not TWEAK-induced Cdc42 activation. Finally, inappropriate expression of Fn14 or Ect2 in mouse astrocytes in vivo using an RCAS vector system for glial-specific gene transfer in G-tva transgenic mice induces astrocyte migration within the brain, corroborating the in vitro importance of the TWEAK-Fn14 signaling cascade in glioblastoma invasion. Our results suggest that the TWEAK-Fn14 signaling axis stimulates glioma cell migration and invasion through two GEF-GTPase signaling units, Ect2-Cdc42 and Trio-Rac1. Components of the Fn14-Rho GEF-Rho GTPase signaling pathway present innovative drug targets for glioma therapy.
Malignant gliomas are the most common primary brain tumors. Despite intensive clinical investigation and significant technical
advances in surgical and radiation treatment, the impact on clinical ...outcome for patients with malignant gliomas is disappointing.
We have previously shown that tumor necrosis factor–like weak inducer of apoptosis (TWEAK), a member of the tumor necrosis
factor superfamily, can stimulate glioma cell survival via binding to the Fn14 receptor, activation of the NF-κB pathway,
and upregulation of BCL-X L gene expression. Here, we show that TWEAK treatment of glioma cells leads to phosphorylation of Akt and BAD. TWEAK stimulation
results in the phosphorylation of both Akt1 and Akt2. However, small interfering RNA (siRNA)–mediated depletion of either
Akt1 or Akt2 showed that BAD serine 136 phosphorylation is dependent specifically on Akt2 function. Depletion of Akt2 expression
by siRNA also abrogates TWEAK-stimulated glioma cell survival, whereas no effect on glioma cell survival was observed after
siRNA-mediated depletion of Akt1 expression. Surprisingly, although siRNA-mediated depletion of BAD in glioma cells abrogates
cytotoxic- and chemotherapy-induced apoptosis, TWEAK still displays a strong protective effect, suggesting that BAD serine
136 phosphorylation plays a minor role in TWEAK-Akt2–induced glioma cell survival. We also report here that AKT2 gene expression levels increased with glioma grade and inversely correlate with patient survival. Additionally, immunohistochemical
analysis showed that Akt2 expression positively correlates with Fn14 expression in glioblastoma multiforme specimens. We hypothesize
that the TWEAK-Fn14 signaling axis functions, in part, to enhance glioblastoma cell survival by activation of the Akt2 serine/threonine
protein kinase. (Mol Cancer Res 2009;7(11):1871–81)
Abstract Glioblastoma remains difficult to treat with immunotherapy due to the immunosuppressive tumor microenvironment and the ability of glioma cells to invade diffusely into normal brain ...parenchyma. While it is known that glioblastoma-associated myeloid cells (GAMs) comprise a large proportion of the tumor microenvironment and can suppress adaptive and innate immune responses through various mechanisms, detailed mapping of myeloid lineages in glioblastoma is still missing. Further characterization of pro-tumoral myeloid-lineages in glioblastoma is important for identifying myeloid-based drug targets that may synergize with T cell-based therapies and produce a more meaningful anti-tumor immune response. Towards this end, we examined numerous scRNAseq datasets derived from glioblastoma patients and identified expression of TNFRSF12A, the gene encoding the TNF- family receptor, Fn14, in a subset of GAMs enriched for expression of cytokines such as CCL3, CCL5, IL-6, and IL-8. This result is intriguing since TWEAK (TNFSF12), the ligand for Fn14, is abundantly expressed within numerous patient-derived glioblastoma cell lines, namely GBM6, GBM8, GBM22, and GBM39. To validate Fn14 protein expression in GAMs, we isolated murine bone-marrow derived macrophages and treated them with glioma-conditioned media, which induced Fn14 gene expression. We then conducted in vitro experiments utilizing the human microglia line HMC3, the murine microglia line SIM-A9, and primary murine bone-marrow-derived macrophages to assess the effects of Fn14 activation in myeloid cells. We found that treatment of Fn14+ myeloid cells with TWEAK ligand upregulates CCL3, CCL5, IL-6, and IL-8 expression. We tested the conditioned media generated from TWEAK-treated Fn14+ myeloid cell lines and found that it promotes glioblastoma invasion in a CCL5-dependent manner. This aligns with previous reports demonstrating the CCL5-CCR5 signaling in glioma cells promotes invasion through calcium-dependent matrix metalloproteinase 2. To assess the spatial distribution of this myeloid subpopulation, we performed RNAscope hybridization against AIF1, TMEM119, CCR5, CCL5, and Fn14 transcripts on sections of murine gliomas generated using the RCAS/tv-a model of human glioblastoma. We found an abundance of Fn14+ plus AIF1+ as well as Fn14+plus TMEM119+ myeloid cells throughout the tumor core and rim of murine tumors and identified CCL5 expression in pseudo-palisades bordering necrotic regions. Our results to date suggest that Fn14 expression in GAMs can promote the invasion of tumor cells into normal brain parenchyma by release of CCL5. Currently, we are utilizing orthotopic patient-derived xenograft models co-implanted with Fn14+/- myeloid cells to test the hypothesis that TWEAK-Fn14 signaling in GAMs provides strong pro-tumoral signals in the glioblastoma microenvironment. Citation Format: Angad Beniwal, Matthew Dufault, Pranjali Kanvinde, David Nascari, Ryan Eghlimi, Adarsha Malla, Dolores Hambardzumyan, Henrique Borges da Silva, Graeme Woodworth, Jeffrey Winkles, Nhan Tran. TWEAK-Fn14 signaling in glioblastoma-associated myeloid cells promotes tumor invasion via CCL5-CCR5 axis abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6867.
Abstract
Glioblastoma multiforme (GBM) is the most malignant primary adult brain tumor. Despite efforts at surgical resection of the glioma mass, invasive cells are always left behind and the tumor ...will inevitably recur and kill the patient. As such, novel therapeutics targeting pro-invasive factors could improve neurological outcomes and survival for these patients. This requires a detailed understanding of the mechanisms driving glioma migration and invasion. We have been investigating whether the multifunctional cytokine tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor fibroblast growth factor-inducible 14 (Fn14) regulate glioma cell invasive activity. We have previously demonstrated that Fn14 expression was elevated in the invasive rim of GBM specimens and migrating glioma cells in vitro. The Fn14 signaling axis is known to drive glioma invasion via Rac1. We have previously reported that Ect2, a guanine nucleotide exchange factor (GEF) for Rho family GTPases, including Rac1 and Cdc42, is overexpressed in GBM, and that overexpression of Ect2 correlates directly with tumor grade and inversely with patient survival. In this study we show that Ect2 regulates Rac1 activation downstream of Fn14. Depletion of Ect2 by siRNA duplexes abrogates Fn14-induced Rac1 activation and subsequent glioma cell migration and invasion. We also found that Cdc42 activation by TWEAK is directly mediated by Ect2. Interestingly, depletion of Cdc42 expression impairs TWEAK-induced Rac1 activation and also results in a significant reduction of glioma cell migration and invasion. This suggests that Cdc42 is, in part, important for Rac1 activation downstream of the TWEAK-Fn14 signaling pathway and argues that another GEF(s) may be involved in the Fn14-Rac1 signaling axis. Recently, we have identified Trio as an additional GEF that activates Rac1 downstream of Cdc42. It is also known that Trio expression correlates directly with brain tumor grade and inversely with patient survival. siRNA-mediated depletion of Trio inhibits TWEAK-induced Rac1 activation but not TWEAK-induced Cdc42 activation. Therefore, delineating the mechanisms of Fn14-RhoGEF-RhoGTPase signaling pathway, may lead to identification of novel targets that can serve as possible points of therapeutic intervention. (Supported by NIH R01-CA130940)
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 5128.
Malignant gliomas are the most common primary brain tumors. Despite intensive clinical investigation and significant technical advances in surgical and radiation treatment, the impact on clinical ...outcome for patients with malignant gliomas is disappointing. We have previously shown that TWEAK, a member of the TNF superfamily, can stimulate glioma cell survival via binding to the Fn14 receptor, activation of the NF-κB pathway, and up-regulation of
BCL-X
L
gene expression. Here, we show that TWEAK treatment of glioma cells leads to phosphorylation of Akt and BAD. TWEAK stimulation results in the phosphorylation of both Akt1 and Akt2. However, siRNA-mediated depletion of either Akt1 or Akt2 showed that BAD serine 136 phosphorylation is dependent specifically on Akt2 function. Depletion of Akt2 expression by siRNA also abrogates TWEAK-stimulated glioma cell survival, whereas no effect on glioma cell survival was observed after siRNA-mediated depletion of Akt1 expression. Surprisingly, although siRNA-mediated depletion of BAD in glioma cells abrogates cytotoxic- and chemotherapy-induced apoptosis, TWEAK still display a strong protective effect, suggesting that BAD serine 136 phosphorylation plays a minor role in TWEAK-Akt2 induced glioma cells survival. We also report here that
AKT2
gene expression levels increased with glioma grade and inversely correlate with patient survival. Additionally, immunohistochemical analysis showed that Akt2 expression positively correlates with Fn14 expression in GBM specimens. We hypothesize that the TWEAK-Fn14 signaling axis functions, in part, to enhance glioblastoma cell survival by activation of the Akt2 serine/threonine protein kinase.
A 60-year-old man was referred as an emergency with a 3 month history of left sided abdominal pain and weight loss. He had no other medical problems and took no medications. An endoscopy was ...performed. This demonstrated a normal oesophagus and stomach and a fleshy mass in the first part of the duodenum with surrounding slough. As this was presumed to be malignant, biopsies were taken. Computed tomography (CT) scan of the abdomen was performed which showed a large, complex, loculated intra-abdominal collection containing air. Results from duodenal biology showed the presence of ulcer slough and liver tissue. The patient was diagnosed with a perforated duodenal ulcer, which had occurred some months previously, and which had eroded into the liver. He was observed and treated with intravenous antibiotics. The patient was discharged on day 14. Follow-up CT scan at 6 weeks showed complete resolution of the collection.
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