Cancer extracellular vesicles (EVs) shuttle at distance and fertilize pre-metastatic niches facilitating subsequent seeding by tumor cells. However, the link between EV secretion mechanisms and their capacity to form pre-metastatic niches remains obscure. Using mouse models, we show that GTPases of the Ral family control, through the phospholipase D1, multi-vesicular bodies homeostasis and tune the biogenesis and secretion of pro-metastatic EVs. Importantly, EVs from RalA or RalB depleted cells have limited organotropic capacities in vivo and are less efficient in promoting metastasis. RalA and RalB reduce the EV levels of the adhesion molecule MCAM/CD146, which favors EV-mediated metastasis by allowing EVs targeting to the lungs. Finally, RalA, RalB and MCAM/CD146, are factors of poor prognosis in breast cancer patients. Altogether, our study identifies RalGTPases as central molecules linking the mechanisms of EVs secretion and cargo loading to their capacity to disseminate and induce pre-metastatic niches in a CD146 dependent manner.
Sequencing and mass spectrometry data have been deposited to EV-Track knowledgebase
Comprehensive molecular portraits of human breast tumoursTCGA breast invasive carcinoma cohort (1097 patients).
- Jacky G Goetz
- Jacky G Goetz
- Jacky G Goetz
- Jacky G Goetz
- Jacky G Goetz
- Christine Carapito
- Nicolas Vitale
- Laetitia Fouillen
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Animal experimentation: All animals were housed and han- dled according to the guidelines of INSERM and the eth- ical committee of Alsace, France (CREMEAS) (Direc- tive 2010/63/EU on the protection of animals used for scientific purposes). Animal facility agreement num- ber: C67-482-33. Experimental license for mice: Apafis 4707-20l6032416407780; experimental license for zebrafish: Apafis 16862-2018121914292754.
Human subjects: Paraffin sections of 4 µm from metastasic and non-metastasic breast tumours were obtained from CRB-Tumorothèque of the Institut de Cancérologie de l'Ouest (ICO, Saint-Herblain, France) (Heymann et al., 2020). Patients were diagnosed and treated at ICO (Integrated Center for Oncology, St Herblain) for metastatic and non-metastatic breast cancers. Samples were processed and included in the CRB-Tumorothèque ICO upon donor agreement and informed consent. Samples and related information are destroyed at the request of the donor. The CRB-Tumorothèque ICO have been declared to and authorized by the French Research Ministry (Declaration Number: DC-2018-3321). This declaration includes approval by a research ethics committee (CPP "Comité de protection des personnes"), in accordance with the French legislation of the Public Health Code.
- Erica A Golemis, Fox Chase Cancer Center, United States
© 2021, Ghoroghi et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Pancreatic cancer is the seventh leading cause of cancer-related death worldwide, and despite advancements in disease management, the 5 -year survival rate stands at only 12%. Triptolides have potent anti-tumor activity against different types of cancers, including pancreatic cancer, however poor solubility and toxicity limit their translation into clinical use. We synthesized a novel pro-drug of triptolide, (E)–19-[(1’-benzoyloxy-1’-phenyl)-methylidene]-Triptolide (CK21), which was formulated into an emulsion for in vitro and in vivo testing in rats and mice, and used human pancreatic cancer cell lines and patient-derived pancreatic tumor organoids. A time-course transcriptomic profiling of tumor organoids treated with CK21 in vitro was conducted to define its mechanism of action, as well as transcriptomic profiling at a single time point post-CK21 administration in vivo. Intravenous administration of emulsified CK21 resulted in the stable release of triptolide, and potent anti-proliferative effects on human pancreatic cancer cell lines and patient-derived pancreatic tumor organoids in vitro, and with minimal toxicity in vivo. Time course transcriptomic profiling of tumor organoids treated with CK21 in vitro revealed <10 differentially expressed genes (DEGs) at 3 hr and ~8,000 DEGs at 12 hr. Overall inhibition of general RNA transcription was observed, and Ingenuity pathway analysis together with functional cellular assays confirmed inhibition of the NF-κB pathway, increased oxidative phosphorylation and mitochondrial dysfunction, leading ultimately to increased reactive oxygen species (ROS) production, reduced B-cell-lymphoma protein 2 (BCL2) expression, and mitochondrial-mediated tumor cell apoptosis. Thus, CK21 is a novel pro-drug of triptolide that exerts potent anti-proliferative effects on human pancreatic tumors by inhibiting the NF-κB pathway, leading ultimately to mitochondrial-mediated tumor cell apoptosis.
The Polycomb Repressive Complex 2 (PRC2) methylates H3K27 to regulate development and cell fate by transcriptional silencing. Alteration of PRC2 is associated with various cancers. Here, we show that mouse Kdm1a deletion causes dramatic reduction of PRC2 proteins, whereas mouse null mutation of L3mbtl3 or Dcaf5 results in PRC2 accumulation and increased H3K27 trimethylation. The catalytic subunit of PRC2, EZH2, is methylated at lysine 20 (K20), promoting EZH2 proteolysis by L3MBTL3 and the CLR4DCAF5 ubiquitin ligase. KDM1A (LSD1) demethylates the methylated K20 to stabilize EZH2. K20 methylation is inhibited by AKT-mediated phosphorylation of serine 21 in EZH2. Mouse Ezh2K20R/K20R mutants develop hepatosplenomegaly associated with high GFI1B expression, and Ezh2K20R/K20R mutant bone marrows expand hematopoietic stem cells and downstream hematopoietic populations. Our studies reveal that EZH2 is regulated by methylation-dependent proteolysis, which is negatively controlled by AKT-mediated S21 phosphorylation to establish a methylation-phosphorylation switch to control the PRC2 activity and hematopoiesis.