Intratumoral hypoxia and expression of Hypoxia Inducible Factor 1α (HIF1α) correlate with metastasis and poor survival in sarcoma patients. We demonstrate here that hypoxia controls sarcoma metastasis through a novel mechanism wherein HIF1α enhances expression of the intracellular enzyme procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2). We show that loss of HIF1α or PLOD2 expression disrupts collagen modification, cell migration and pulmonary metastasis (but not primary tumor growth) in allograft and autochthonous LSLKrasG12D/+; Trp53fl/fl murine sarcoma models. Furthermore, ectopic PLOD2 expression restores migration and metastatic potential in HIF1α-deficient tumors, and analysis of human sarcomas reveal elevated HIF1α and PLOD2 expression in metastatic primary lesions. Pharmacological inhibition of PLOD enzymatic activity suppresses metastases. Collectively, these data indicate that HIF1α controls sarcoma metastasis through PLOD2-dependent collagen modification and organization in primary tumors. We conclude that PLOD2 is a novel therapeutic target in sarcomas and successful inhibition of this enzyme may reduce tumor cell dissemination.
Genetic aberrations responsible for soft-tissue sarcoma formation in adults are largely unknown, with targeted therapies sorely needed for this complex and heterogeneous family of diseases. Here we report that that the Hippo pathway is deregulated in many soft-tissue sarcomas, resulting in elevated expression of the effector molecule Yes-Associated Protein (YAP). Based on data gathered from human sarcoma patients, a novel autochthonous mouse model, and mechanistic analyses, we determined that YAP-dependent expression of the transcription factor forkhead box M1 (FOXM1) is necessary for cell proliferation/tumorigenesis in a subset of soft-tissue sarcomas. Notably, FOXM1 directly interacts with the YAP transcriptional complex via TEAD1, resulting in coregulation of numerous critical pro-proliferation targets that enhance sarcoma progression. Finally, pharmacologic inhibition of FOXM1 decreases tumor size in vivo, making FOXM1 an attractive therapeutic target for the treatment of some sarcoma subtypes.
Cancer cells are characterized by rapid proliferation and require adaptive metabolic responses to allow continued biosynthesis and cell growth in the setting of decreased oxygen (O 2 ) and nutrient availability. The hypoxia-inducible factors (HIFs) are a common link between adaptation to low O 2 , changes in cancer metabolism, and malignant progression. The HIF-a subunits differentially regulate metabolic enzymes and other key factors involved in glycolysis, changes in redox status, and oxidative phosphorylation. Importantly, metabolic changes can, in turn, regulate HIF activity. Finally, changes in metabolism under hypoxia lead to important crosstalk between cancer cells and the stromal compartment of the microenvironment.Keywords Hypoxia Á HIF Á Cancer metabolism Á Tumor microenvironment Hypoxia-inducible factors and their regulationA majority of healthy tissues experience 2-9 % O 2 , while hypoxia is defined as less than 2 % O 2 [1]. Low O 2 tensions are often exhibited by regions of intense inflammation such as within arthritic joints, regions of the bone marrow, and in highly proliferative cancer cells [2]. Hypoxia may occur secondary to necrosis or aberrant neovascularization resulting in poor perfusion. Additionally, cancer cells may also proliferate rapidly enough to outstrip their blood supply [3]. Cells adapt to changes in O 2 availability by altering gene expression of crucial metabolic enzymes in order to counter changes in nutrient availability and redox status. This response is mediated, in part, by O 2 -labile transcription factors hypoxia-inducible factors HIF-1a and HIF-2a, key regulators of cellular adaptation to hypoxic stress [3][4][5].Comprised of an O 2 -sensitive a subunit and constitutively expressed b subunit, HIFs are primarily regulated through post-translational modification and stabilization and are part of the basic helix-loop-helix-PAS (bHLH/ PAS) family of transcription factors [6]. Under normal O 2 tensions, prolyl hydroxylase domain enzymes (PHDs) hydroxylate two conserved proline residues (405 and 531 in HIF-1a) within the O 2 -dependent degradation (ODD) domain of the HIF-a subunit. After hydroxylation, the von Hippel-Lindau (VHL) tumor suppressor E3 ligase complex polyubiquitinates HIF-a and targets it for eventual degradation by the 26S proteasome [7][8][9]. Under low O 2 , HIFs are no longer modified by PHDs, but instead dimerize with ARNT/HIF-1b through HLH and PAS domain interactions, translocate to the nucleus, and recruit coactivators such as CBP/p300. HIF heterodimers bind and recognize hypoxiaresponse elements (HREs), with the consensus sequence G/ACGTG, within the promoter regions of target genes and drive adaptive gene transcription [10][11][12][13] (Fig. 1). While the HIF-1a subunit is expressed ubiquitously, HIF-2a is selectively expressed in a much more tissue-restricted manner but can be found at high levels in vascular endothelial cells and myeloid-derived cells [14].V. Mucaj and J.E.S. Shay contributed equally to this work.
Glioblastoma multiforme (GBM) and the mesenchymal GBM subtype in particular are highly malignant tumors that frequently exhibit regions of severe hypoxia and necrosis. Because these features correlate with poor prognosis, we investigated microRNAs whose expression might regulate hypoxic GBM cell survival and growth. We determined that the expression of microRNA-218 (miR-218) is decreased significantly in highly necrotic mesenchymal GBM, and orthotopic tumor studies revealed that reduced miR-218 levels confer GBM resistance to chemotherapy. Importantly, miR-218 targets multiple components of receptor tyrosine kinase (RTK) signaling pathways, and miR-218 repression increases the abundance and activity of multiple RTK effectors. This elevated RTK signaling also promotes the activation of hypoxia-inducible factor (HIF), most notably HIF2α. We further show that RTK-mediated HIF2α regulation is JNK dependent, via jun proto-oncogene. Collectively, our results identify an miR-218-RTK-HIF2α signaling axis that promotes GBM cell survival and tumor angiogenesis, particularly in necrotic mesenchymal tumors.
Hypoxia-inducible factors (HIFs) accumulate in both neoplastic and inflammatory cells within the tumor microenvironment and impact the progression of a variety of diseases, including colorectal cancer. Pharmacological HIF inhibition represents a novel therapeutic strategy for cancer treatment. We show here that acriflavine (ACF), a naturally occurring compound known to repress HIF transcriptional activity, halts the progression of an autochthonous model of established colitis-associated colon cancer (CAC) in immunocompetent mice. ACF treatment resulted in decreased tumor number, size and advancement (based on histopathological scoring) of CAC. Moreover, ACF treatment corresponded with decreased macrophage infiltration and vascularity in colorectal tumors. Importantly, ACF treatment inhibited the hypoxic induction of M-CSFR, as well as the expression of the angiogenic factor (vascular endothelial growth factor), a canonical HIF target, with little to no impact on the Nuclear factor-kappa B pathway in bone marrow-derived macrophages. These effects probably explain the observed in vivo phenotypes. Finally, an allograft tumor model further confirmed that ACF treatment inhibits tumor growth through HIF-dependent mechanisms. These results suggest pharmacological HIF inhibition in multiple cell types, including epithelial and innate immune cells, significantly limits tumor growth and progression.
Glioblastomas are aggressive adult brain tumors, characterized by inadequately organized vasculature and consequent nutrient and oxygen (O2)-depleted areas. Adaptation to low nutrients and hypoxia supports glioblastoma cell survival, progression, and therapeutic resistance. However, specific mechanisms promoting cellular survival under nutrient and O2 deprivation remain incompletely understood. Here, we show that miR-124 expression is negatively correlated with a hypoxic gene signature in glioblastoma patient samples, suggesting that low miR-124 levels contribute to pro-survival adaptive pathways in this disease. Since miR-124 expression is repressed in various cancers (including glioblastoma), we quantified miR-124 abundance in normoxic and hypoxic regions in glioblastoma patient tissue, and investigated whether ectopic miR-124 expression compromises cell survival, during tumor ischemia. Our results indicate that miR-124 levels are further diminished in hypoxic/ischemic regions within individual glioblastoma patient samples, compared to regions replete in O2 and nutrients. Importantly, we also show that increased miR-124 expression affects the ability of tumor cells to survive under O2 and/or nutrient deprivation. Moreover, miR-124 re-expression increases cell death in vivo, and enhances the survival of mice bearing intracranial xenograft tumors. miR-124 exerts this phenotype in part by directly regulating TEAD1, MAPK14/p38α and SERP1, factors involved in cell proliferation and survival under stress. Simultaneous suppression of these miR-124 targets results in similar levels of cell death as caused by miR-124 restoration. Importantly, we further demonstrate that SERP1 re-introduction reverses the hypoxic cell death elicited by miR-124, indicating the importance of SERP1 in promoting tumor cell survival. In support of our experimental data, we observed a significant correlation between high SERP1 levels and poor patient outcome in glioblastoma patients. Collectively, among the many pro-tumorigeneic properties of miR-124 repression in glioblastoma, we delineated a novel role in promoting tumor cell survival under stressful microenvironments, thereby supporting tumor progression.
Receptor tyrosine kinase (RTK) pathway signaling plays a central role in the growth and progression of Glioblastoma (GBM), a highly aggressive group of brain tumors. We recently reported that miR-218 repression, an essentially uniform feature of human GBM, directly promotes RTK hyperactivation by increasing the expression of key positive signaling effectors, including EGFR, PLCγ1, PIK3CA and ARAF (1). However, enhanced RTK signaling is known to activate compensatory inhibitory feedback mechanisms in both normal and cancer cells. We demonstrate here that miR-218 repression in GBM cells also increases the abundance of additional upstream and downstream signaling mediators, including PDGFRα, RSK2, and S6K1, which collectively function to alleviate inhibitory RTK feedback regulation. In turn, RTK signaling suppresses miR-218 expression via STAT3, which binds directly to the miR-218 locus, along with BCLAF1, to repress its expression. These data identify novel interacting feedback loops by which miR-218 repression promotes increased RTK signaling in high-grade gliomas.
In the urgent setting of the COVID-19 pandemic, treatment hypotheses abound, each of which requires careful evaluation. A randomized controlled trial generally provides the strongest possible evaluation of a treatment, but the efficiency and effectiveness of the trial depend on the existing evidence supporting the treatment. The researcher must therefore compile a body of evidence justifying the use of time and resources to further investigate a treatment hypothesis in a trial. An observational study can help provide this evidence, but the lack of randomized exposure and the researcher’s inability to control treatment administration and data collection introduce significant challenges for nonexperimental studies. A proper analysis of observational health care data thus requires an extensive background in a diverse set of topics ranging from epidemiology and causal analysis to relevant medical specialties and data sources. Here we provide 10 rules that serve as an end-to-end introduction to retrospective analyses of observational health care data. A running example of a COVID-19 study presents a practical implementation of each rule in the context of a specific treatment hypothesis. When carefully designed and properly executed, a retrospective analysis framed around these rules will inform the decisions of whether and how to investigate a treatment hypothesis in a randomized controlled trial.
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