Glioblastoma is a malignant brain tumor that portends a poor prognosis. Its resilience, in part, is related to a remarkable capacity for manipulating the microenvironment to promote its growth and ...survival. Microglia/macrophages are prime targets, being drawn into the tumor and stimulated to produce factors that support tumor growth and evasion from the immune system. Here we show that the RNA regulator, HuR, plays a key role in the tumor-promoting response of microglia/macrophages. Knockout (KO) of HuR led to reduced tumor growth and proliferation associated with prolonged survival in a murine model of glioblastoma. Analysis of tumor composition by flow cytometry showed that tumor associated macrophages (TAMs) were decreased, more polarized toward an M1-like phenotype, and had reduced PD-L1 expression. There was an overall increase in infiltrating CD4
+
cells, including Th1 and cytotoxic effector cells, and a concomitant reduction in tumor-associated polymorphonuclear myeloid-derived suppressor cells. Molecular and cellular analyses of HuR KO TAMs and cultured microglia showed changes in migration, chemoattraction, and chemokine/cytokine profiles that provide potential mechanisms for the altered tumor microenvironment and reduced tumor growth in HuR KO mice. In summary, HuR is a key modulator of pro-glioma responses by microglia/macrophages through the molecular regulation of chemokines, cytokines, and other factors. Our findings underscore the relevance of HuR as a therapeutic target in glioblastoma.
Abstract
Development of effective novel anti-tumor treatments will require improved in vitro models that incorporate physiologic microenvironments and maintain intratumoral heterogeneity including ...tumor initiating cells. Brain tumor initiating cells (BTIC) are a target for cancer therapy because they are highly tumorigenic and contribute to tumor angiogenesis, invasion, and therapeutic resistance. Current leading studies rely on BTIC isolation from patient-derived xenografts followed by propagation as neurospheres. As this process is expensive and time-consuming, we determined whether three-dimensional microtumors were an alternative in vitro method for modeling tumor growth via BITC maintenance and/or enrichment. Brain tumor cells were grown as neurospheres or as microtumors produced using a human-derived biomatrix HuBiogelTM and maintained with physiologically relevant microenvironments. Percentages of BITCs were determined based on cell surface marker expression (CD133), label retention (carboxyfluorescein succinimidyl ester; CFSE), and tumorsphere formation capacity. Our data demonstrate that expansion of brain tumor cells as hypoxic and nutrient restricted microtumors significantly increased the percentage of both CD133+ and CFSE+ cells. We further demonstrate that BTIC-marker positive cells isolated from microtumors maintain neurosphere formation capacity in the in vitro limiting dilution assay and tumorigenic potential in vivo. These data demonstrate that microtumors can be a useful three-dimensional biological model for the study of BTIC maintenance and targeting.
Citation Format: Ashley Gilbert, Kiera Walker, Anh Tran, Yancey Gillespie, Raj Singh, Anita B. Hjelmeland. Modeling physiologic microenvironments in three-dimensional microtumors facilitates brain tumor initiating cell maintenance abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1925. doi:10.1158/1538-7445.AM2017-1925
Endothelial cell dysfunction and apoptosis are critical in the pathogenesis of atherosclerotic cardiovascular disease (CVD). Both endothelial cell apoptosis and atherosclerosis are reduced by ...high-density lipoprotein (HDL). Low HDL levels increase the risk of CVD and are also a key characteristic of the metabolic syndrome. The apolipoprotein E4 (APOE4) allele also increases the risk of atherosclerosis and CVD. We previously demonstrated that the antiapoptotic activity of HDL is inhibited by APOE4 very-low-density lipoprotein (APOE4-VLDL) in endothelial cells, an effect similar to reducing the levels of HDL. Here we establish the intracellular mechanism by which APOE4-VLDL inhibits the antiapoptotic pathway activated by HDL. We show that APOE4-VLDL diminishes the phosphorylation of Akt by HDL but does not alter phosphorylation of c-Jun N-terminal kinase, p38, or Src family kinases by HDL. Furthermore APOE4-VLDL inhibits Akt phosphorylation by reducing the phosphatidylinositol 3-kinase product phosphatidylinositol-(3,4,5)-triphosphate (PI3,4,5P3). We further demonstrate that APOE4-VLDL reduces PI(3,4,5)P3, through the phosphoinositol phosphatase SHIP2, and not through PTEN. SHIP2 is already implicated as an independent risk factor for type II diabetes, hypertension and obesity, which are also all components of the metabolic syndrome and independent risk factors for CVD. Significantly, the association between CVD and type 2 diabetes or hypertension is further increased by the APOE4 allele. Therefore the activation of SHIP2 by APOE4-VLDL, with the subsequent inhibition of the HDL/Akt pathway, is a novel and significant biological mechanism and may be a critical intermediate by which APOE4 increases the risk of atherosclerotic CVD.
Abstract
Brain tumor initiating cells (BTICs) are resistant to chemo- and radiotherapy, providing a reservoir for tumor recurrence and a desirable target for glioma treatments. Standard of care for ...glioblastoma (GBM; grade IV astrocytoma) includes the chemotherapeutic agent temozolomide, which prolongs life expectancy by months and is not curative. Prior studies suggested the efficacy of chemotherapies including temozolomide was increased by reducing expression of carbonic anhydrase 9 (CA9). CA9 is a hypoxia responsive gene elevated in tumors that is important for regulating intracellular pH and contributing to the acidic extracellular microenvironment. After confirming basal and hypoxia-induced expression of CA9 in GBM BTICs, we targeted CA9 activity with the small molecule inhibitor SLC-0111 alone or in combination with temozolomide. In multiple GBM BTIC lines, SLC-0111 reduced cell growth in vitro and showed additional benefit when used concurrently with temozolomide. Importantly, SLC-0111 inhibited the enrichment of BTICs after temozolomide treatment as determined via BTIC marker expression and neurosphere formation capacity. These data suggested the potential of SLC-0111 as a chemosensitizer, which we next evaluated in preclinical studies using a subcutaneous recurrent GBM model. GBMs treated with SLC-0111 in combination with temozolomide significantly regressed and the resulting in vivo growth delay was greater than that of temozolomide or SLC-0111 alone. Together, our data suggest that SLC-0111 can sensitize GBM BTICs to the chemotherapy temozolomide and significantly delay disease progression.
Citation Format: Nathaniel H. Boyd, Kiera Walker, Paul C. McDonald, Mark O. Bevensee, Yancey G. Gillespie, Burt Nabors, Shoukat Dedhar, Anita B. Hjelmeland. Targeting the tumor microenvironment: inhibition of carbonic anhydrase 9 impedes brain tumor initiating cell chemoresistance and delays glioblastoma growth in vivo abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 171. doi:10.1158/1538-7445.AM2017-171
Abstract
Background: Glioblastoma (GBM) is the most common malignant primary brain tumor with very poor prognosis. The current standard of care for GBM consists of maximal safe tumor resection, ...chemotherapy (temozolomide) and radiotherapy followed by adjuvant temozolomide and Tumor Treating Fields (TTFields). Unfortunately, GBM displays both inherent and acquired resistance to temozolomide and radiation and is incurable. While TTFields have been found to broadly induce endoplasmic reticulum stress, autophagy, cell membrane permeability, DNA replication stress, anti-mitotic and anti-migratory effects, it is unclear how well TTFields impact radiation resistant GBM. To better understand the cytotoxic benefit of TTFields on acquired radiation resistant GBM, we tested patient-derived xenograft (PDX) brain tumor initiating cells (BTICs) that had been selected for radiation resistance and compared them to their radiation sensitive parent.
Methods: PDX were developed with subcutaneous implantation of patient GBM cells into immunodeficient (nu/nu) mice. Acquired radiation resistant PDXs were generated through in vivo selection (6-8 passages) using six fractions of 2 Gray of radiation over two weeks. PDX BTICs were cultured on laminin-coated cover slips under serum free conditions. Cytotoxicity was measured on both the two-dimensional sham control and two-dimensional TTFields treatment conditions over the course of 72 hours and analyzed with crystal violet staining for both imaging and absorbance quantification. PamStation 12 Kinomic data and RNA-seq (Illumina) data was generated for each PDX.
Results: Crystal violet quantification and representative imaging indicated that TTFields inhibit cell growth in both parental and acquired radiation resistant PDX-derived BTICs. There is an observed TTFields sensitivity response from the acquired radiation resistant PDX, indicating that TTFields treatment does have a cytotoxic effect on acquired radiation resistant models (seen over four reproducible experiments). Baseline kinomic and transcriptomic differences between the PDX lines suggest potential TTFields sensitivity signatures.
Conclusions: Ongoing studies are focused on validation of this cytotoxic effect on larger cohort of both acquired and inherent radiation resistant PDX cells. We plan to measure TTFields -treatment induced kinomic alterations to identify downstream signaling pathways associated with TTFields treatment response, as well as the development of acquired TTFields resistant GBM PDX cells.
Citation Format: Taylor L. Schanel, Amber B. Jones, Andee M. Beierle, Hasan Alrefai, Lauren C. Nassour, Patricia H. Hicks, Joshua C. Anderson, Anita B. Hjelmeland, Christopher D. Willey. Establishing the cytotoxic benefit of Tumor Treating Fields on radiation sensitive and acquired radiation resistant glioblastoma patient derived xenograft pairs abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1102.
Abstract
Glioblastoma (GBM), or grade IV astrocytoma, is a deadly disease due in part to the high degree of intratumoral heterogeneity that contributes to treatment failures. Previous studies have ...shown the importance of reactive species balances, partially controlled by the coupling of nitric oxide synthases (NOS) with their cofactor, in maintenance of glioma stem cell (GSC) phenotype as well as survival of cancer cells in general. In this study, we investigated the roles of GTP cyclohydrolase 1 (GCH1), which is the first and rate-limiting enzyme of the pathway producing of NOS cofactor producing pathway, in GBM stem cell phenotypes via redox balances. We found that GCH1 RNA and protein expression were increased in GSCs in comparison to non-GSCs, but that GCH1 was not exclusive to the GSC fraction. Indeed, GCH1 was elevated in GBM in comparison to normal brain. Overexpression of GCH1 in GBM cells increased cell growth in vitro and neurosphere-forming capability and decreased survival in an intracranial GBM mouse model. In contrast, GCH1 knockdown with short hairpin RNA in GBM cells led to growth inhibition in vitro as well as increased survival in animal models. GCH1 increased CD44 expression and was upregulated in the detrimental mesenchymal GBM subtype in which CD44 served as a marker. Mechanistically, we found that the expression of GCH1 increased BH4 production, as well as augmented multiple antioxidant pathways, including the expression of PARK7, was critical for controlling reactive species balance, including suppressing reactive oxygen species production. In silico analyses demonstrated that higher GCH1 levels in glioma patients correlate with higher glioma grade, recurrence and worse survival. Together, our data suggest that upregulation of GCH1 in GSCs promotes tumor maintenance and is a key regulator of reactive oxygen species in GBM, and GCH1 pathway is a potential target for therapy.
Citation Format: Anh N. Tran, Kiera Walker, David G. Harrison, Wei Chen, James Mobley, Lauren Hocevar, James R. Hackney, Randee Sedaka, Jennifer Pollock, Matthew S. Goldberg, Dolores Hambardzumyan, Sara J. Cooper, G Yancey Gillespie, Anita B. Hjelmeland. Glioblastoma, cancer stem cells, and reactive species balances: A case for GTP cyclohydrolase 1 abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 163.
Telomerase is an important mechanism by which cancers escape replicative senescence. In neural tumors, cancer stem cells express telomerase suggesting that this may explain their preferential ...tumorigenesis. Oligonucleotide telomerase targeting selectively disrupts cancer stem cell growth through the induction of differentiation, adding to the armamentarium of anti-cancer stem cell therapies.
Tumorigenic and non-neoplastic tissue injury occurs via the ischemic microenvironment defined by low oxygen, pH, and nutrients due to blood supply malfunction. Ischemic conditions exist within ...regions of pseudopalisading necrosis, a pathological hallmark of glioblastoma (GBM), the most common primary malignant brain tumor in adults. To recapitulate the physiologic microenvironment found in GBM tumors and tissue injury, we developed an in vitro ischemic model and identified chromodomain helicase DNA binding protein 7 (CHD7) as a novel ischemia-regulated gene. Point mutations in the CHD7 gene are causal in CHARGE syndrome, a CNS developmental disorder, and interrupt the epigenetic functions of CHD7 in regulating neural stem cell maintenance and development. Using our ischemic system, we observed microenvironment-mediated decreases in CHD7 expression in brain tumor initiating cells and neural stem cells. Validating our approach, CHD7 was suppressed in the perinecrotic niche of GBM patient and xenograft sections, and an interrogation of patient gene expression datasets determined correlations between low CHD7, increasing glioma grade and worse patient outcomes. Segregation of GBM by molecular subtype revealed a novel observation that CHD7 expression is elevated in proneural vs mesenchymal GBM. Genetic targeting of CHD7 and subsequent gene ontology analysis of RNA sequencing data indicated angiogenesis as a primary biological function affected by CHD7 expression changes. We validated this finding in tube formation assays and vessel formation in orthotopic GBM models. Together, our data provide further understanding of molecular responses to ischemia and a novel function of CHD7 in regulating angiogenesis in both neoplastic and non-neoplastic systems.
The ischemic microenvironment in both tumor and non-neoplastic tissue can drive new blood vessel formation to adapt to changes in pH, oxygen tension, and restricted nutrient availability. Using neural progenitor and glioblastoma cells in an in vitro ischemic model, we demonstrated that mRNA and protein expression of the epigenetic reader CHD7 was suppressed in the ischemic microenvironment. Reducing CHD7 altered the transcriptome to increase angiogenesis-related pathways that include CHI3L1 (YKL-40) and increased measures of angiogenesis in vitro and in vivo.
Abstract
Glioblastomas (GBMs) are the most common primary brain tumors in adults and one of the most aggressive cancers with high rates of recurrence and therapeutic resistance. In GBMs, ...subpopulations of highly tumorigenic cells called brain tumor initiating cells (BTICs) have been characterized by their unique capacity to promote tumor maintenance, therapeutic resistance, and angiogenesis. BTIC maintenance is known to be regulated by reactive oxygen and nitrogen species. GTP cyclohydrolase, or GCH1, is a critical molecule regulating reactive species levels. We found that expression of GCH1 RNA and protein were upregulated in BTICs in comparison to non-BTICs. Overexpression of GCH1 in glioma cells increased cell growth in vitro and increased tumor formation and decreased survival in an intracranial GBM mouse model. In contrast, GCH1 depletion with short hairpin RNA in GBM cells led to growth inhibition in vitro as well as increased survival in animal orthotopic models. Furthermore, genetic modulation of GCH1 led to altered ROS levels in GBM xenolines. In silico analyses demonstrate that higher GCH1 levels in glioma patients correlate with higher glioma grade, recurrence and worse survival. Together, our data suggest that upregulation of GCH1 in BTICs promotes tumor maintenance and is a key regulator of reactive oxygen species in glioblastoma.
Citation Format: Anh N. Tran, Kiera Walker, David Harisson, Wei Chen, James Mobley, Lauren Hocevar, James R. Hackney, Randee S. Sedaka, Jennifer S. Pollock, Sara J. Cooper, George Y. Gillespie, Anita B. Hjelmeland. GTP cyclohydrolase in brain tumor stem cells is implicated in glioblastoma growth abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5462. doi:10.1158/1538-7445.AM2017-5462
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