Despite a growing body of knowledge about the genomic landscape and molecular pathogenesis of sarcomas, translation of basic discoveries into targeted therapies and significant clinical gains has ...remained elusive. Renewed interest in altered metabolic properties of cancer cells has led to an exploration of targeting metabolic dependencies as a novel therapeutic strategy. In this study, we have characterized the dependency of human pediatric sarcoma cells on key metabolic substrates and identified a mechanism of adaptation to metabolic stress by examining proliferation and bioenergetic properties of rhabdomyosarcoma and Ewing sarcoma cells under varying concentrations of glucose and glutamine. While all cell lines tested were completely growth-inhibited by lack of glucose, cells adapted to glutamine deprivation, and restored proliferation following an initial period of reduced growth. We show that expression of glutamine synthetase (GS), the enzyme responsible for de novo glutamine synthesis, increased during glutamine deprivation, and that pharmacological or shRNA-mediated GS inhibition abolished proliferation of glutamine-deprived cells, while having no effect on cells grown under normal culture conditions. Moreover, the GS substrates and glutamine precursors glutamate and ammonia restored proliferation of glutamine-deprived cells in a GS-dependent manner, further emphasizing the necessity of GS for adaptation to glutamine stress. Furthermore, pharmacological and shRNA-mediated GS inhibition significantly reduced orthotopic xenograft tumor growth. We also show that glutamine supports sarcoma nucleotide biosynthesis and optimal mitochondrial bioenergetics. Our findings demonstrate that GS mediates proliferation of glutamine-deprived pediatric sarcomas, and suggest that targeting metabolic dependencies of sarcomas should be further investigated as a potential therapeutic strategy.
Altered cellular metabolism, including an increased dependence on aerobic glycolysis, is a hallmark of cancer. Despite the fact that this observation was first made nearly a century ago, effective ...therapeutic targeting of glycolysis in cancer has remained elusive. One potentially promising approach involves targeting the glycolytic enzyme lactate dehydrogenase (LDH), which is overexpressed and plays a critical role in several cancers. Here, we used a novel class of LDH inhibitors to demonstrate, for the first time, that Ewing sarcoma cells are exquisitely sensitive to inhibition of LDH. EWS-FLI1, the oncogenic driver of Ewing sarcoma, regulated LDH A (LDHA) expression. Genetic depletion of LDHA inhibited proliferation of Ewing sarcoma cells and induced apoptosis, phenocopying pharmacologic inhibition of LDH. LDH inhibitors affected Ewing sarcoma cell viability both
and
by reducing glycolysis. Intravenous administration of LDH inhibitors resulted in the greatest intratumoral drug accumulation, inducing tumor cell death and reducing tumor growth. The major dose-limiting toxicity observed was hemolysis, indicating that a narrow therapeutic window exists for these compounds. Taken together, these data suggest that targeting glycolysis through inhibition of LDH should be further investigated as a potential therapeutic approach for cancers such as Ewing sarcoma that exhibit oncogene-dependent expression of LDH and increased glycolysis. SIGNIFICANCE: LDHA is a pharmacologically tractable EWS-FLI1 transcriptional target that regulates the glycolytic dependence of Ewing sarcoma.
Breast cancer (BC) can recur as metastatic disease many years after primary tumor removal, suggesting that disseminated tumor cells survive for extended periods in a dormant state that is refractory ...to conventional therapies. We have previously shown that altering the tumor microenvironment through fibrosis with collagen and fibronectin deposition can trigger tumor cells to switch from a dormant to a proliferative state. Here, we used an in vivo preclinical model and a 3D in vitro model of dormancy to evaluate the role of Src family kinase (SFK) in regulating this dormant-to-proliferative switch. We found that pharmacological inhibition of SFK signaling or Src knockdown results in the nuclear localization of cyclin-dependent kinase inhibitor p27 and prevents the proliferative outbreak of dormant BC cells and metastatic lesion formation; however, SFK inhibition did not kill dormant cells. Dormant cell proliferation also required ERK1/2 activation. Combination treatment of cells undergoing the dormant-to-proliferative switch with the Src inhibitor (AZD0530) and MEK1/2 inhibitor (AZD6244) induced apoptosis in a large fraction of the dormant cells and delayed metastatic outgrowth, neither of which was observed with either inhibitor alone. Thus, targeting Src prevents the proliferative response of dormant cells to external stimuli, but requires MEK1/2 inhibition to suppress their survival. These data indicate that treatments targeting Src in combination with MEK1/2 may prevent BC recurrence.
Embryonal rhabdomyosarcoma (ERMS) is a childhood cancer that expresses myogenic master regulatory factor MYOD but fails to differentiate. Here, we show that the zinc finger transcription factor CASZ1 ...up-regulates MYOD signature genes and induces skeletal muscle differentiation in normal myoblasts and ERMS. The oncogenic activation of the RAS-MEK pathway suppresses CASZ1 expression in ERMS. ChIP-seq, ATAC-seq and RNA-seq experiments reveal that CASZ1 directly up-regulates skeletal muscle genes and represses non-muscle genes through affecting regional epigenetic modifications, chromatin accessibility and super-enhancer establishment. Next generation sequencing of primary RMS tumors identified a single nucleotide variant in the CASZ1 coding region that potentially contributes to ERMS tumorigenesis. Taken together, loss of CASZ1 activity, due to RAS-MEK signaling or genetic alteration, impairs ERMS differentiation, contributing to RMS tumorigenesis.
Metastasis, a complex process, is responsible for most deaths in patients with cancer. Clinically relevant research models are indispensable to advancing our understanding of metastatic mechanisms ...and developing new treatments. We here describe detailed protocols to establish mouse models for melanoma metastasis using the single-cell imaging system and orthotropic footpad injection. The single-cell imaging system permits the tracking and quantification of early metastatic cell survival, while the orthotropic footpad transplantation mimics aspects of the complex metastatic process.
For complete details on the use and execution of this protocol, please refer to Yu et al.1,2
Display omitted
•The single-cell imaging system for tracking metastatic cell survival in vivo•Tail vein injection for establishing experimental melanoma metastasis model•The orthotropic footpad injection for building up spontaneous melanoma metastasis model
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
Metastasis, a complex process, is responsible for most deaths in patients with cancer. Clinically relevant research models are indispensable to advancing our understanding of metastatic mechanisms and developing new treatments. We here describe detailed protocols to establish mouse models for melanoma metastasis using the single-cell imaging system and orthotropic footpad injection. The single-cell imaging system permits the tracking and quantification of early metastatic cell survival, while the orthotropic footpad transplantation mimics aspects of the complex metastatic process.
Background
Chromosomal translocations generating oncogenic transcription factors are the hallmark of a variety of tumors, including many sarcomas. Ewing sarcoma family of tumors (ESFTs) are ...characterized by the t(11;22)(q24;q12) translocation that generates the Ewing sarcoma breakpoint region 1 and Friend leukemia virus integration 1 (EWS-FLI1) fusion transcription factor responsible for the highly malignant phenotype of this tumor. Although continued expression of EWS-FLI1 is believed to be critical for ESFT cell survival, a clinically effective small-molecule inhibitor remains elusive likely because EWS-FLI1 is a transcription factor and therefore widely felt to be "undruggable."
Methods
We developed a high-throughput screen to evaluate more than 50 000 compounds for inhibition of EWS-FLI1 activity in TC32 ESFT cells. We used a TC32 cell-based luciferase reporter screen using the EWS-FLI1 downstream target NR0B1 promoter and a gene signature secondary screen to sort and prioritize the compounds. We characterized the lead compound, mithramycin, based on its ability to inhibit EWS-FLI1 activity in vitro using microarray expression profiling, quantitative reverse transcription-polymerase chain reaction, and immunoblot analysis, and in vivo using immunohistochemistry. We studied the impact of this inhibition on cell viability in vitro and on tumor growth in ESFT xenograft models in vivo (n = 15-20 mice per group). All statistical tests were two-sided.
Results
Mithramycin inhibited expression of EWS-FLI1 downstream targets at the mRNA and protein levels and decreased the growth of ESFT cells at half maximal inhibitory concentrations between 10 (95% confidence interval CI = 8 to 13 nM) and 15 nM (95% CI = 13 to 19 nM). Mithramycin suppressed the growth of two different ESFT xenograft tumors and prolonged the survival of ESFT xenograft-bearing mice by causing a decrease in mean tumor volume. For example, in the TC32 xenograft model, on day 15 of treatment, the mean tumor volume for the mithramycin-treated mice was approximately 3% of the tumor volume observed in the control mice (mithramycin vs control: 69 vs 2388 mm3, difference = 2319 mm3, 95% CI = 1766 to 2872 mm3, P < .001).
Conclusion
Mithramycin inhibits EWS-FLI1 activity and demonstrates ESFT antitumor activity both in vitro and in vivo.
Abstract The insulin-like growth factor 1 receptor (IGF-1R) has surfaced as a significant target in multiple solid cancers due to its fundamental roles in pro-survival and anti-apoptotic signaling. ...However, development of resistance to IGF-1R blockade represents a significant hindrance and limits treatment efficacy in the clinic. In this study, we identified acquired resistance to IGF-1R blockade with R1507, an antibody against IGF-1R, and with BMS-754807, a small molecular inhibitor of IGF-1R/insulin receptor (IR). We showed that treatment with an IGF-IR antibody, R1507, or an IR/IGF-IR kinase inhibitor, BMS-754807, was associated with increased activation of YES/SRC family tyrosine kinase (SFK) in rhabdomyosarcoma (RMS). Combining anti–IGF-1R agents with SFK inhibitors resulted in blockade of IGF-1R inhibition–induced activation of YES/SFK and displayed advantageous antitumor activity in vitro and in vivo . Our data provide evidence that IGF-1R blockade results in activation of the YES/SRC family kinase bypass resistance pathway in vitro and in vivo . This may be of particular clinical relevance since both Yes and IGF components are overexpressed in RMS. Increased YES/SFK activation might serve as a clinical biomarker for predicting tumor resistance to IGF-1R inhibition. Dual inhibition of IGF-1R and SFK may have a broader and enhanced clinical benefit for patients with RMS.
Although many cancers are showing remarkable responses to targeted therapies, pediatric sarcomas, including Ewing sarcoma, remain recalcitrant. To broaden the therapeutic landscape, we explored the
...response of Ewing sarcoma cell lines against a large collection of investigational and approved drugs to identify candidate combinations.
Drugs displaying activity as single agents were evaluated in combinatorial (matrix) format to identify highly active, synergistic drug combinations, and combinations were subsequently validated in multiple cell lines using various agents from each class. Comprehensive metabolomic and proteomic profiling was performed to better understand the mechanism underlying the synergy. Xenograft experiments were performed to determine efficacy and
mechanism.
Several promising candidates emerged, including the combination of small-molecule PARP and nicotinamide phosphoribosyltransferase (NAMPT) inhibitors, a rational combination as NAMPTis block the rate-limiting enzyme in the production of nicotinamide adenine dinucleotide (NAD
), a necessary substrate of PARP. Mechanistic drivers of the synergistic cell killing phenotype of these combined drugs included depletion of NMN and NAD
, diminished PAR activity, increased DNA damage, and apoptosis. Combination PARPis and NAMPTis
resulted in tumor regression, delayed disease progression, and increased survival.
These studies highlight the potential of these drugs as a possible therapeutic option in treating patients with Ewing sarcoma.
.
Abstract Metastasis is the cause of more than 90% of all cancer deaths. Despite this fact, most anticancer therapeutics currently in clinical use have limited efficacy in treating established ...metastases. Here, we identify the endoplasmic reticulum chaperone protein, glucose-regulated protein 78 (GRP78), as a metastatic dependency in several highly metastatic cancer cell models. We find that GRP78 is consistently upregulated when highly metastatic cancer cells colonize the lung microenvironment and that mitigation of GRP78 upregulation via short hairpin RNA or treatment with the small molecule IT-139, which is currently under clinical investigation for the treatment of primary tumors, inhibits metastatic growth in the lung microenvironment. Inhibition of GRP78 upregulation and an associated reduction in metastatic potential have been shown in four highly metastatic cell line models: three human osteosarcomas and one murine mammary adenocarcinoma. Lastly, we show that downmodulation of GRP78 in highly metastatic cancer cells significantly increases median survival times in our in vivo animal model of experimental metastasis. Collectively, our data indicate that GRP78 is an attractive target for the development of antimetastatic therapies.
PDF
