Cancer metabolism, including in mitochondria, is a disease hallmark and therapeutic target, but its regulation is poorly understood. Here, we show that many human tumors have heterogeneous and often ...reduced levels of Mic60, or Mitofilin, an essential scaffold of mitochondrial structure. Despite a catastrophic collapse of mitochondrial integrity, loss of bioenergetics, and oxidative damage, tumors with Mic60 depletion slow down cell proliferation, evade cell death, and activate a nuclear gene expression program of innate immunity and cytokine/chemokine signaling. In turn, this induces epithelial-mesenchymal transition (EMT), activates tumor cell movements through exaggerated mitochondrial dynamics, and promotes metastatic dissemination in vivo. In a small-molecule drug screen, compensatory activation of stress response (GCN2) and survival (Akt) signaling maintains the viability of Mic60-low tumors and provides a selective therapeutic vulnerability. These data demonstrate that acutely damaged, "ghost" mitochondria drive tumor progression and expose an actionable therapeutic target in metastasis-prone cancers.
Treatment for the lethal primary adult brain tumor glioblastoma (GBM) includes the chemotherapy temozolomide (TMZ), but TMZ resistance is common and correlates with promoter methylation of the DNA ...repair enzyme O-6-methylguanine-DNA methyltransferase (MGMT). To improve treatment of GBMs, including those resistant to TMZ, we explored the potential of targeting dopamine receptor signaling. We found that dopamine receptor 3 (DRD3) is expressed in GBM and is also a previously unexplored target for therapy. We identified novel antagonists of DRD3 that decreased the growth of GBM xenograft-derived neurosphere cultures with minimal toxicity against human astrocytes and/or induced pluripotent stem cell-derived neurons. Among a set of DRD3 antagonists, we identified two compounds, SRI-21979 and SRI-30052, that were brain penetrant and displayed a favorable therapeutic window analysis of The Cancer Genome Atlas data demonstrated that higher levels of DRD3 (but not DRD2 or DRD4) were associated with worse prognosis in primary, MGMT unmethylated tumors. These data suggested that DRD3 antagonists may remain efficacious in TMZ-resistant GBMs. Indeed, SRI-21979, but not haloperidol, significantly reduced the growth of TMZ-resistant GBM cells. Together our data suggest that DRD3 antagonist-based therapies may provide a novel therapeutic option for the treatment of GBM.
The multifaceted roles of metabolism in invasion have been investigated across many cancers. The brain tumor glioblastoma (GBM) is a highly invasive and metabolically plastic tumor with an inevitable ...recurrence. The neuronal glucose transporter 3 (GLUT3) was previously reported to correlate with poor glioma patient survival and be upregulated in GBM cells to promote therapeutic resistance and survival under restricted glucose conditions. It has been suggested that the increased glucose uptake mediated by GLUT3 elevation promotes survival of circulating tumor cells to facilitate metastasis. Here we suggest a more direct role for GLUT3 in promoting invasion that is not dependent upon changes in cell survival or metabolism. Analysis of glioma datasets demonstrated that GLUT3, but not GLUT1, expression was elevated in invasive disease. In human xenograft derived GBM cells, GLUT3, but not GLUT1, elevation significantly increased invasion in transwell assays, but not growth or migration. Further, there were no changes in glycolytic metabolism that correlated with invasive phenotypes. We identified the GLUT3 C-terminus as mediating invasion: substituting the C-terminus of GLUT1 for that of GLUT3 reduced invasion. RNA-seq analysis indicated changes in extracellular matrix organization in GLUT3 overexpressing cells, including upregulation of osteopontin. Together, our data suggest a role for GLUT3 in increasing tumor cell invasion that is not recapitulated by GLUT1, is separate from its role in metabolism and survival as a glucose transporter, and is likely broadly applicable since GLUT3 expression correlates with metastasis in many solid tumors.
Tumor heterogeneity has hampered the development of novel effective therapeutic options for aggressive cancers, including the deadly primary adult brain tumor glioblastoma (GBM). Intratumoral ...heterogeneity is partially attributed to the tumor initiating cell (TIC) subset that contains highly tumorigenic, stem-like cells. TICs display metabolic plasticity but can have a reliance on aerobic glycolysis. Elevated expression of GLUT1 and GLUT3 is present in many cancer types, with GLUT3 being preferentially expressed in brain TICs (BTICs) to increase survival in low nutrient tumor microenvironments, leading to tumor maintenance. Through structure-based virtual screening (SBVS), we identified potential novel GLUT inhibitors. The screening of 13 compounds identified two that preferentially inhibit the growth of GBM cells with minimal toxicity to non-neoplastic astrocytes and neurons. These compounds, SRI-37683 and SRI-37684, also inhibit glucose uptake and decrease the glycolytic capacity and glycolytic reserve capacity of GBM patient-derived xenograft (PDX) cells in glycolytic stress test assays. Our results suggest a potential new therapeutic avenue to target metabolic reprogramming for the treatment of GBM, as well as other tumor types, and the identified novel inhibitors provide an excellent starting point for further lead development.
De-regulated cellular energetics is an emerging hallmark of cancer with alterations to glycolysis, oxidative phosphorylation, the pentose phosphate pathway, lipid oxidation and synthesis and amino ...acid metabolism. Understanding and targeting of metabolic reprogramming in cancers may yield new treatment options, but metabolic heterogeneity and plasticity complicate this strategy. One highly heterogeneous cancer for which current treatments ultimately fail is the deadly brain tumor glioblastoma. Therapeutic resistance, within glioblastoma and other solid tumors, is thought to be linked to subsets of tumor initiating cells, also known as cancer stem cells. Recent profiling of glioblastoma and brain tumor initiating cells reveals changes in metabolism, as compiled here, that may be more broadly applicable. We will summarize the profound role for metabolism in tumor progression and therapeutic resistance and discuss current approaches to target glioma metabolism to improve standard of care.
Abstract
Glioblastoma (GBM) is the most common, lethal primary adult brain tumor with patient survival of only 14 months. The location and invasion of GBM leads to rapid recurrence after therapy. The ...standard of care chemotherapy is DNA damaging agent temozolomide (TMZ), to which resistance is common and is due, partly, to expression of the DNA repair enzyme O-6-methylguanine-DNA methyltransferase(MGMT), regulated by promoter methylation. To improve treatment of GBMs, including those resistant to TMZ, we explored targeting dopamine receptor signaling. Prior reports indicated roles for dopamine receptor 2 and 4 in GBM, with these inhibitors being effective in combination with EGFR inhibitors or temozolomide, respectively. We demonstrate that dopamine receptor 3 (DRD3) is an alternative target for therapy, with an expected low risk of severe side effects due to restricted expression in non-tumor brain. Six novel antagonists of DRD3 decreased the growth of GBM xenograft-derived neurosphere cultures, with minimal toxicity against human astrocytes and neurons. For those compounds, with a potential therapeutic window, two (SRI-21979 and SRI-30052) readily crossed the BBB and yielded no signs of liver or kidney dysfunction. In orthotopic models, 10 mg/kg of SRI-21979 per day for ten days, alone or combined with TMZ, trends toward increased survival. In striking contrast, we observed no benefit for haloperidol treatment in combination with TMZ beyond that for TMZ alone. Further analysis of TCGA data demonstrated that, unlike DRD2 and DRD4, DRD3 levels were not reduced in MGMT unmethylated GBMs and higher levels of DRD3 were associated with worse prognosis, suggesting that DRD3 antagonists may remain efficacious in TMZ resistant GBMs. SRI-21979, but not haloperidol or TMZ, significantly reduced the growth of TMZ resistant U251 cells and neurospheres derived from a TMZ-resistant xenograft. Our data demonstrate that DRD3 antagonist based combinatorial therapies may provide a potential, novel therapeutic treatment for GBM.
Abstract
Dysregulated sphingolipid metabolism is associated with many cancers; allowing cells to evade apoptosis through increases in sphingosine-1-phosphate (S1P) and decreases in ceramides. ...Ceramides can be hydrolyzed by ceramidases to sphingosine, which can then be phosphorylated by sphingosine kinases to S1P. S1P allows cells to evade apoptosis and increase migration, while shifts toward ceramides favor cell death. Glioblastoma (GBM) exhibits shifts in the sphingolipid balance towards S1P, contributing to chemoresistance and migration. Understanding of sphingolipid metabolism in GBM is still limited, and currently, there are no approved treatments to target the dysregulation. Acid ceramidase (ASAH1), a key enzyme in the production of S1P, is highly expressed in GBM and is associated with worse survival of GBM patients, as per The Cancer Genome Atlas data. To address the altered sphingolipid metabolism and therapeutic resistance in GBM, we explored the efficacy of pharmacologic and genetic inhibition of ASAH1 in both parental and temozolomide (TMZ)-resistant patient-derived xenografts. Cells were infected with ASAH1 shRNA or treated with ASAH1 inhibitors and assessed for cell growth and migration. Our work suggests that pharmacologic inhibition of ASAH1 induces cell death and that this effect is maintained in TMZ-resistant cells. Furthermore, we find a novel role for carmofur, an ASAH1 inhibitor, in the inhibition of GBM migration. Together, these data suggest the potential utility of normalizing the sphingolipid balance in the context of GBM TMZ resistance.
Abstract
Despite available treatments including surgical resection, radiation and chemotherapy, glioblastoma (GBM) is incurable with rapid recurrence and low median survival rate of just fourteen ...months. Development of more effective treatments is difficult due to the highly heterogeneous nature of GBM. One aspect of that heterogeneity involves brain tumor initiating cells (BTICs) that have a stem cell-like ability to self-renew. BTICs can readily alter their metabolism and survive in low nutrient environments due in part to increased GLUT3 expression. We believe that the higher expression of GLUT3 in cancer cells compared to non-tumor cells makes it a therapeutic target, although the potential for toxicity must be considered. In recently accepted studies by Libby et al., we reported on two novel GLUT inhibitors identified by structure based virtual screening (SBVS) using a GLUT3 homology model. We are creating a structure-activity relationship profile and seek to increase the potency, selectivity and stability of the GLUT inhibitors. In this study we have tested a number of novel analogs and identified three that have maintained efficacy against BTICs in vitro. Importantly, these compounds display minimal toxicity against human astrocytes. The novel derivatives have increased stability compared to the lead compounds and are efficacious in the nanomolar range. In the future, we intend to utilize our anti-GLUT compounds alone and in combination with radio- and chemotherapy with the hope of clinical translation.
Glioblastoma (GBM) is the most common adult primary malignant brain tumor with a median survival of about 15 months, even after aggressive treatment. Treatment of GBM is difficult for multiple ...reasons including the location of the tumor, tumor invasiveness, and the high degree of both inter-and intra-tumoral heterogeneity. Contributing to intratumoral heterogeneity are highly tumorigenic, stem-like tumor cells, with the capacity to self-renew and propagate the tumor, termed brain tumor initiating cells (BTICs). BTICs are also commonly therapy resistant, highly invasive, and metabolically plastic with elevated expression of glucose transporter 3 (GLUT3) allowing them to preferentially survive in low nutrient microenvironments. GLUT3 correlated with poor patient prognosis in GBM, a segregation not seen with GLUT1, another glucose transporter isoform highly expressed in cancers. Data from the literature indicated that GLUT3 correlated with metastasis in several solid tumors and we found elevated GLUT3 expression at the GBM invasive edge. We therefore investigated the role of GLUT3 in invasion using overexpression as a model system. Overexpression of GLUT3 promoted GBM PDX invasion both in vitro and in vivo, a phenotype not seen with GLUT1 overexpression. Generating chimeric proteins in which regions of low homology between GLUT1 and GLUT3 were swapped, we demonstrated a role for the C-terminus in the pro-invasive GLUT3 phenotype. Considering the importance of GLUTs in the glycolytic shift of GBM cells, the restricted expression of GLUT3 in non-tumor tissues, the role of GLUT3 in BTIC survival and the correlation of GLUT3 with poor patient prognosis, GLUT3 is a promising target for novel anti-GBM therapy development. Using structure based virtual screening we predicted compounds likely to bind GLUT3 that were then assessed for their ability to inhibit both glucose uptake and growth of GBM cells derived from human xenografts. From the first screen, two compounds were identified as leads with the ability to decrease glucose uptake and GBM growth. We then performed a second screen which identified improved compounds with an IC50 of 300-500 nM, but additional studies are needed to improve the efficacy, stability, solubility, and specificity of these compounds so they could ultimately be moved toward the clinic.
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