Small extracellular vesicles (SEVs) are functional messengers of certain cellular niches to permit non-contact cell communications. Whether niche-specific SEVs fulfill this role in cancer is unclear. Here, we used seven cell-type specific mouse Cre lines to conditionally knockout Vps33b in Cdh5+ or Tie2+ endothelial cells (ECs), Lepr+ bone marrow perivascular cells, Osx+ osteo-progenitor cells (OPCs), Pf4+ megakaryocytes and Tcf21+ spleen stromal cells. We then examined the effects of reduced SEV secretion on progression of MLL-AF9 induced acute myeloid leukemia (AML) as well as normal hematopoiesis. Blocking SEV secretion from ECs, but not perivascular cells, megakaryocytes or spleen stromal cells, markedly delayed the leukemia progression. Notably, reducing SEV production from ECs had no effect on normal hematopoiesis. Protein analysis showed that EC-derived SEVs contained a high level of ANGPTL2, which accelerated leukemia progression via binding to LILRB2 receptor. Moreover, ANGPTL2-SEVs released from ECs were governed by VPS33B. Importantly, ANGPTL2-SEVs were also required for primary human AML cell maintenance. These findings demonstrate a role of niche-specific SEVs in cancer development and suggest that targeting ANGPTL2-SEVs from ECs might be a potential strategy to interfere certain types of AML.
Dan Huang, Guohuan Sun, Xiaoxin Hao, Xiaoxiao He, Zhaofeng Zheng, Chiqi Chen, Zhuo Yu, Li Xie, Shihui Ma, Ligen Liu, Bo O. Zhou, Hui Cheng, Junke Zheng, Tao Cheng
Zeb1, a zinc finger E-box binding homeobox epithelial-mesenchymal (EMT) transcription factor, confers properties of ‘stemness’, such as self-renewal, in cancer. Yet little is known about the function of Zeb1 in adult stem cells. Here, we used the hematopoietic system, as a well-established paradigm of stem cell biology, to evaluate Zeb1 mediated regulation of adult stem cells. We employed a conditional genetic approach using the Mx1-Cre system to specifically knockout (KO) Zeb1 in adult hematopoietic stem cells (HSCs) and their downstream progeny. Acute genetic deletion of Zeb1 led to rapid onset thymic atrophy and apoptosis driven loss of thymocytes and T cells. A profound cell-autonomous self-renewal defect and multi-lineage differentiation block was observed in Zeb1 KO HSCs. Loss of Zeb1 in HSCs activated transcriptional programs of deregulated HSC maintenance and multi-lineage differentiation genes, and of cell polarity, consisting of cytoskeleton, lipid metabolism/lipid membrane and cell adhesion related genes. Notably, Epithelial cell adhesion molecule (EpCAM) expression was prodigiously upregulated in Zeb1 KO HSCs, which correlated with enhanced cell survival, diminished mitochondrial metabolism, ribosome biogenesis, and differentiation capacity and an activated transcriptomic signature associated with acute myeloid leukemia (AML) signaling. ZEB1 expression was downregulated in AML patients and Zeb1 KO in the malignant counterparts of HSCs - leukemic stem cells (LSCs) - accelerated MLL-AF9 and Meis1a/Hoxa9-driven AML progression, implicating Zeb1 as a tumor suppressor in AML LSCs. Thus, Zeb1 acts as a transcriptional regulator in hematopoiesis, critically co-ordinating HSC self-renewal, apoptotic and multi-lineage differentiation fates required to suppress leukemic potential in AML.
Alhomidi Almotiri, Hamed Ahmad A. Alzahrani, Juan Bautista Menendez-Gonzalez, Ali Abdelfattah, Badi Alotaibi, Lubaid Saleh, Adelle Greene, Mia R. F. Georgiou, Alex Gibbs, Amani Salem Alsayari, Sarab Taha, Leigh-Anne Thomas, Dhruv Shah, Sarah Edkins, Peter J. Giles, Marc P. Stemmler, Simone Brabletz, Thomas Brabletz, Ashleigh S. Boyd, Florian A. Siebzehnrubl, Neil P. Rodrigues
Microglia maintain homeostasis in the brain. However, with age, they become primed and respond more strongly to inflammatory stimuli. We show here that microglia from aged mice upregulated mammalian target of rapamycin (mTOR) complex 1 signaling regulating translation, as well as protein levels of inflammatory mediators. Genetic ablation of mTOR signaling showed a dual, yet contrasting effect on microglia priming: it caused an NF-kB-dependent upregulation of priming genes at mRNA level; however, mice displayed reduced cytokine protein levels, diminished microglia activation and milder sickness behavior. The effect on translation was dependent on reduced phosphorylation of 4EBP1, resulting in decreased binding of eIF4E to eIF4G. Similar changes were present in aged human microglia and in damage-associated microglia, indicating upregulation of mTOR-dependent translation is an essential step licensing microglia priming in aging and neurodegeneration.
Lily Keane, Ignazio Antignano, Sean-Patrick Riechers, Raphael Zollinger, Anaelle A. Dumas, Nina Offermann, Maria E. Bernis, Jenny Russ, Frederike J. Graelmann, Patrick N. McCormick, Julia Esser, Dario Tejera, Ai Nagano, Jun Wang, Claude Chelala, Yvonne Biederbick, Annett Halle, Paolo Salomoni, Michael Thomas Heneka, Melania Capasso
Neurofibromatosis type 1 (NF1) is a common tumor predisposition syndrome, caused by NF1 gene mutation, in which affected patients develop Schwann cell lineage peripheral nerve sheath tumors (neurofibromas). To investigate human neurofibroma pathogenesis, we differentiated a series of isogenic patient-specific NF1-mutant human induced-pluripotent stem cells (hiPSCs) into Schwannian lineage cells (SLCs). We found that while wild-type and heterozygous NF1-mutant hiPSC-SLCs did not form tumors following mouse sciatic nerve implantation, NF1-null SLCs formed bona fide neurofibromas with high levels of SOX10 expression. To confirm that SOX10+ SLCs contain the cells of origin for neurofibromas, both Nf1 alleles were inactivated in mouse Sox10+ cells, leading to classic nodular cutaneous and plexiform neurofibroma formation that completely recapitulate their human counterparts. Moreover, we discovered that NF1 loss impaired Schwann cell differentiation by inducing a persistent stem-like state to expand the pool of progenitors required to initiate tumor formation, indicating that in addition to regulating MAPK-mediated cell growth, NF1 loss also alters Schwann cell differentiation to promote neurofibroma development. Taken together, we established complementary humanized neurofibroma explant and first-in-kind mouse genetically engineered nodular cutaneous neurofibroma models that delineate neurofibroma pathogenesis amenable to future therapeutic target discovery and evaluation.
Juan Mo, Corina Anastasaki, Zhiguo Chen, Tracey Shipman, Jason B. Papke, Kevin Y. Yin, David H. Gutmann, Lu Q. Le
Emerging evidence indicates that early life events can increase the risk for developing chronic obstructive pulmonary disease (COPD). Using an inducible transgenic mouse model for NF-κB activation in the airway epithelium, we found that a brief period of inflammation during the saccular stage [postnatal day (PN)3 - PN5] but not alveolar stage (PN10 - PN12) of lung development disrupts elastic fiber assembly, resulting in permanent reduction in lung function and development of a COPD-like lung phenotype that progresses through 24 months of age. Neutrophil depletion prevented disruption of elastic fiber assembly and restored normal lung development. Mechanistic studies uncovered a role for neutrophil elastase (NE) in downregulating expression of critical elastic fiber assembly components, particularly fibulin-5 and elastin. Further, both purified human NE and NE-containing exosomes from tracheal aspirates of premature infants with lung inflammation down-regulated elastin and fibulin-5 expression by saccular stage mouse lung fibroblasts. Together, our studies define a critical developmental window for assembling the elastin scaffold in the distal lung, which is required to support lung structure and function throughout the lifespan. While neutrophils play a well-recognized role in COPD development in adults, neutrophilic inflammation may also contribute to early life predisposition to COPD.
John T. Benjamin, Erin Plosa, Jennifer Sucre, Riet van der Meer, Shivangi Dave, Sergey S. Gutor, David Nichols, Peter Gulleman, Christopher Jetter, Wei Han, Matthew K. Xin, Peter C. Dinella, Ashley Catanzarite, Seunghyi Kook, Kalsang Dolma, Charitharth V. Lal, Amit Gaggar, J. Edwin Blalock, Dawn C. Newcomb, Bradley W. Richmond, Jonathan A. Kropski, Lisa R. Young, Susan Guttentag, Timothy S. Blackwell
Graft-versus-host disease (GVHD) causes failed reconstitution of donor plasmacytoid dendritic cells (pDCs) that are critical for immune protection and tolerance. We used both murine and human systems to uncover the mechanisms whereby GVHD induces donor pDC defects. GVHD depleted Flt3-expressing donor multipotent progenitors (MPPs) that sustained pDCs, leading to impaired generation of pDCs. MPP loss was associated with decreased amounts of MPP-producing hematopoietic stem cells (HSCs) and oxidative stress-induced death of proliferating MPPs. Additionally, alloreactive T cells produced GM-CSF to inhibit MPP expression of Tcf4, the transcription factor essential for pDC development, subverting MPP production of pDCs. GM-CSF did not affect the maturation of pDC precursors. Notably, enhanced recovery of donor pDCs upon adoptive transfer early after allogeneic HSC transplantation repressed GVHD and restored the de novo generation of donor pDCs in recipient mice. pDCs suppressed the proliferation and expansion of activated autologous T cells via a type-I IFN signaling-dependent mechanism. They also produced PD-L1 and LILRB4 to inhibit T cell production of IFN-. We thus demonstrate that GVHD impairs the reconstitution of tolerogenic donor pDCs by depleting DC progenitors rather than by preventing pDC maturation. MPPs are an important target to effectively bolster pDC reconstitution for controlling GVHD.
Yuanyuan Tian, Lijun Meng, Ying Wang, Bohan Li, Hongshuang Yu, Yan Zhou, Tien Bui, Alicia Li, Ciril Abraham, Yongping Zhang, Jian Wang, Chenchen Zhao, Shin Mineishi, Stefania Gallucci, David Porter, Elizabeth Hexner, Hong Zheng, Yanyun Zhang, Shaoyan Hu, Yi Zhang
Membrane protrusion and adhesion to the extracellular matrix, which involves the extension of actin filaments and formation of adhesion complexes, are the fundamental processes for cell migration, tumor invasion, and metastasis. How cancer cells efficiently coordinate these processes remains unclear. Here, we showed that membrane-targeted CLIC1 spatiotemporally regulates the formation of cell-matrix adhesions and membrane protrusions through the recruitment of PIP5Ks to the plasma membrane. Comparative proteomics identified CLIC1 upregulated in human hepatocellular carcinoma (HCC) and associated with tumor invasiveness, metastasis, and poor prognosis. In response to migration-related stimuli, CLIC1 recruited PIP5K1A and PIP5K1C from the cytoplasm to the leading edge of the plasma membrane, where PIP5Ks generate a PIP2-rich microdomain to induce the formation of integrin-mediated cell-matrix adhesions and the signaling for cytoskeleon extension. CLIC1 silencing inhibited the attachment of tumor cells to culture plates and the adherence and extravasation in the lung alveoli resulting in suppressed lung metastasis in mice. This study reveals an unrecognized mechanism that spatiotemporally coordinates the formation of both lamellipodium/invadopodia and nascent cell-matrix adhesions for directional migration and tumor invasion/metastasis. The unique traits of upregulation and membrane targeting of CLIC1 in cancer cells make it an excellent therapeutic target for tumor metastasis.
Jei-Ming Peng, Sheng-Hsuan Lin, Ming-Chin Yu, Sen-Yung Hsieh
MYC stimulates both metabolism and protein synthesis, but it is unknown how cells coordinate these complementary programs. Previous work reported that in a subset of small cell lung cancer (SCLC) cell lines, MYC activates guanosine triphosphate (GTP) synthesis and results in sensitivity to inhibitors of the GTP synthesis enzyme inosine monophosphate dehydrogenase (IMPDH). Here we demonstrated that primary MYCHigh human SCLC tumors also contain abundant guanosine nucleotides. We also found that elevated MYC in SCLCs with acquired chemoresistance rendered these otherwise recalcitrant tumors dependent on IMPDH. Unexpectedly, our data indicated that IMPDH links the metabolic and protein synthesis outputs of oncogenic MYC. Co-expression analysis placed IMPDH within the MYC-driven ribosome program, and GTP depletion prevented RNA Polymerase I (Pol I) from localizing to ribosomal DNA. Furthermore, the GTPases GPN1 and GPN3 were upregulated by MYC and directed Pol I to ribosomal DNA. Constitutively GTP-bound GPN1/3 mutants mitigated the effect of GTP depletion on Pol I, protecting chemoresistant SCLC cells from IMPDH inhibition. GTP therefore functions as a metabolic gate tethering MYC-dependent ribosome biogenesis to nucleotide sufficiency through GPN1 and GPN3. IMPDH dependence is a targetable vulnerability in chemoresistant, MYCHigh SCLC.
Fang Huang, Kenneth Huffman, Zixi Wang, Xun Wang, Kailong Li, Feng Cai, Chendong Yang, Ling Cai, Terry S. Shih, Lauren G. Zacharias, Andrew S. Chung, Qian Yang, Milind D. Chalishazar, Abbie S. Ireland, C. Allison Stewart, Kasey R. Cargill, Luc Girard, Yi Liu, Min Ni, Jian Xu, Xudong Wu, Hao Zhu, Benjamin J. Drapkin, Lauren A. Byers, Trudy G. Oliver, Adi Gazdar, John Minna, Ralph DeBerardinis
While platelets are the cellular mediators of thrombosis, platelets are also immune cells. Platelets interact both directly and indirectly with immune cells, impacting their activation and differentiation, as well as all phases of the immune response. Megakaryocytes (Mks) are the cell source of circulating platelets, and until recently Mks were typically only considered as bone marrow (BM) resident cells. However, platelet producing Mks also reside in the lung, and lung Mks express greater levels of immune molecules compared to BM Mks. We therefore sought to define the immune functions of lung Mks. Using single cell RNA-Seq of BM and lung myeloid enriched cells, we found that lung Mks (MkL) had gene expression patterns that are similar to antigen presenting cells (APC). This was confirmed using imaging and conventional flow cytometry. The immune phenotype of Mks was plastic and driven by the tissue immune environment as evidenced by BM Mks having a MkL like phenotype under the influence of pathogen receptor challenge and lung associated immune molecules, such as IL-33. Our in vitro and in vivo assays demonstrated that MkL internalized and processed both antigenic proteins and bacterial pathogens. Furthermore, MkL induced CD4+ T cell activation in a MHC II dependent manner both in vitro and in vivo. These data indicated that Mks in the lung had key immune regulatory roles dictated in part by the tissue environment.
Daphne N. Pariser, Zachary T. Hilt, Sara K. Ture, Sara K. Blick-Nitko, Mark R. Looney, Simon J. Cleary, Estheany Roman-Pagan, Jerry Saunders II, Steve N. Georas, Janelle M. Veazey, Ferralita Madere, Laura Tesoro Santos, Allison M. Arne, Nguyen PT Huynh, Alison C. Livada, Selena M. Guerrero-Martin, Claire E. Lyons, Kelly A. Metcalf Pate, Kathleen E. McGrath, James Palis, Craig Morrell
Psoriasis is a chronic inflammatory skin disease characterized by inflammatory cell infiltration, as well as hyperproliferation of keratinocytes in skin lesions, and is considered a metabolic syndrome. We found that the expression of galectin-7 is reduced in the skin lesions of patients with psoriasis. IL-17A and TNF-α, two cytokines intimately involved in the development of psoriatic lesions, suppressed galectin-7 expression in human primary keratinocytes (HEKn cells) and the immortalized human keratinocyte cell line HaCaT. A galectin-7 knockdown in these cells elevated the production of IL-6 and IL-8 and enhanced ERK signaling when the cells were stimulated with IL-17A. Galectin-7 attenuated IL-17A–induced production of inflammatory mediators by keratinocytes via the miR-146a–ERK pathway. Moreover, galectin-7–deficient mice showed enhanced epidermal hyperplasia and skin inflammation in response to intradermal IL-23 injection. We identified fluvastatin as an inducer of galectin-7 expression by connectivity map (cMAP) analysis, confirmed this effect in keratinocytes, and demonstrated that fluvastatin attenuated IL-6 and IL-8 production induced by IL-17A. Thus, we validate a role of galectin-7 in the pathogenesis of psoriasis, in both epidermal hyperplasia and keratinocyte-mediated inflammatory responses, and formulated a rationale for the use of statins in the treatment of psoriasis.
Hung-Lin Chen, Chia-Hui Lo, Chi-Chun Huang, Meng-Ping Lu, Po-Yuan Hu, Chang-Shan Chen, Di-Yen Chueh, Peilin Chen, Teng-Nan Lin, Yuan-Hsin Lo, Yu-Ping Hsiao, Daniel K. Hsu, Fu-Tong Liu
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