Reduced nuclear lamin A/C enhances cancer cell squeezing through rigid barriers, does not facilitate endothelial crossing, and impairs experimental metastasis
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE151176 | GEO | 2021/06/02
REPOSITORIES: GEO
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Project description:Reduced nuclear lamin A/C enhances cancer cell squeezing through rigid barriers, does not facilitate endothelial crossing, and impairs experimental metastasis
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Project description:The mechanisms by which the nuclear lamina of tumor cells influences tumor growth and migration are highly disputed. Lamin A and its variant lamin C are key lamina proteins that control nucleus stiffness and chromatin conformation. Downregulation of lamin A/C in two prototypic metastatic lines, B16F10 melanoma and E0771 breast carcinoma, facilitated cell squeezing through rigid pores, and reduced heterochromatin content. Surprisingly, both lamin A/C knockdown cells grew poorly in 3D spheroids within soft agar, and lamin A/C deficient cells derived from spheroids transcribed lower levels of the growth regulator Yap1. Unexpectedly, the transendothelial migration of both cancer cells in vitro and in vivo, through lung capillaries, was not elevated by lamin A/C knockdown and their metastasis in lungs was even dramatically reduced. Our results are the first indication that reduced lamin A/C content in distinct types of highly metastatic cancer cells does not elevate their transendothelial migration (TEM) capacity and diapedesis through lung vessels but can compromise lung metastasis at a post extravasation level.
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Project description:Tissues can be soft like fat, which bears little stress, or stiff like bone, which sustains high stress, but whether there is a systematic relationship between tissue mechanics and differentiation is unknown. Here, proteomics analyses revealed that levels of the nucleoskeletal protein lamin-A scaled with tissue elasticity, E, as did levels of collagens in the extracellular matrix that determine E. Stem cell differentiation into fat on soft matrix was enhanced by low lamin-A levels, whereas differentiation into bone on stiff matrix was enhanced by high lamin-A levels. Matrix stiffness directly influenced lamin-A protein levels, and, although lamin-A transcription was regulated by the vitamin A/retinoic acid (RA) pathway with broad roles in development, nuclear entry of RA receptors was modulated by lamin-A protein. Tissue stiffness and stress thus increase lamin-A levels, which stabilize the nucleus while also contributing to lineage determination.
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Project description:Nuclear lamins are important structural and functional proteins in mammalian cells, but little is known about the mechanisms and cofactors that regulate their traffic into the nucleus. Here, we demonstrate that trafficking of lamin A, but not lamin B1, and its assembly into the nuclear envelope are regulated by sorting nexin 6 (SNX6), a major component of the retromer that targets proteins and other molecules to specific subcellular locations. SNX6 interacts with lamin A in vitro and in vivo and links it to the outer surface of the endoplasmic reticulum in human and mouse cells. SNX6 transports its lamin A cargo to the nuclear envelope in a process that takes several hours. Lamin A protein levels in the nucleus augment or decrease, respectively, upon gain or loss of SNX6 function. We further show that SNX6-dependent lamin A nuclear import occurs across the nuclear pore complex via a RAN-GTP-dependent mechanism. These results identify SNX6 as a key regulator of lamin A synthesis and incorporation into the nuclear envelope.
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Project description:Metastasis is the major cause of death in cancer patients. Circulating tumor cells need to migrate through the endothelial layer of blood vessels to escape the hostile circulation and establish metastases at distant organ sites. Here, we identified the membrane-bound metalloprotease ADAM17 on endothelial cells as a key driver of metastasis. We show that TNFR1-dependent tumor cell-induced endothelial cell death, tumor cell extravasation, and subsequent metastatic seeding is dependent on the activity of endothelial ADAM17. Moreover, we reveal that ADAM17-mediated TNFR1 ectodomain shedding and subsequent processing by the γ-secretase complex is required for the induction of TNF-induced necroptosis. Consequently, genetic ablation of ADAM17 in endothelial cells as well as short-term pharmacological inhibition of ADAM17 prevents long-term metastases formation in the lung. Thus, our data identified ADAM17 as a novel essential regulator of necroptosis and as a new promising target for antimetastatic and advanced-stage cancer therapies.
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Project description:Cancer-secreted microRNAs (miRNAs) are emerging mediators of cancer-host crosstalk. Here we show that miR-105, which is characteristically expressed and secreted by metastatic breast cancer cells, is a potent regulator of migration through targeting the tight junction protein ZO-1. In endothelial monolayers, exosome-mediated transfer of cancer-secreted miR-105 efficiently destroys tight junctions and the integrity of these natural barriers against metastasis. Overexpression of miR-105 in nonmetastatic cancer cells induces metastasis and vascular permeability in distant organs, whereas inhibition of miR-105 in highly metastatic tumors alleviates these effects. miR-105 can be detected in the circulation at the premetastatic stage, and its levels in the blood and tumor are associated with ZO-1 expression and metastatic progression in early-stage breast cancer.
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Project description:Transendothelial migration (TEM) of lymphocytes and neutrophils is associated with the ability of their deformable nuclei to displace endothelial cytoskeletal barriers. Lamin A is a key intermediate filament component of the nuclear lamina that is downregulated during granulopoiesis. When elevated, lamin A restricts nuclear squeezing through rigid confinements. To determine if the low lamin A expression by leukocyte nuclei is critical for their exceptional squeezing ability through endothelial barriers, we overexpressed this protein in granulocyte-like differentiated HL-60 cells. A 10-fold higher lamin A expression did not interfere with chemokinetic motility of these granulocytes on immobilized CXCL1. Furthermore, these lamin A high leukocytes exhibited normal chemotaxis toward CXCL1 determined in large pore transwell barriers, but poorly squeezed through 3 μm pores toward identical CXCL1 gradients. Strikingly, however, these leukocytes successfully completed paracellular TEM across inflamed endothelial monolayers under shear flow, albeit with a small delay in nuclear squeezing into their sub-endothelial pseudopodia. In contrast, CXCR2 mediated granulocyte motility through collagen I barriers was dramatically delayed by lamin A overexpression due to a failure of lamin A high nuclei to translocate into the pseudopodia of the granulocytes. Collectively, our data predict that leukocytes maintain a low lamin A content in their nuclear lamina in order to optimize squeezing through extracellular collagen barriers but can tolerate high lamin A content when crossing the highly adaptable barriers presented by the endothelial cytoskeleton.
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Project description:Liver colonization is initiated through the interplay between tumor cells and adhesion molecules present in liver sinusoidal endothelial cells (LSECs). This crosstalk stimulates tumor COX-2 upregulation and PGE2 secretion. To elucidate the role of the LSEC intercellular adhesion molecule-1 (ICAM-1) in the prometastatic response exerted by tumor and stromal COX-2, we utilized celecoxib (CLX) as a COX-2 inhibitory agent. We analyzed the in vitro proliferative and secretory responses of murine C26 colorectal cancer (CRC) cells to soluble ICAM-1 (sICAM-1), cultured alone or with LSECs, and their effect on LSEC and hepatic stellate cell (HSC) migration and in vivo liver metastasis. CLX reduced sICAM-1-stimulated COX-2 activation and PGE2 secretion in C26 cells cultured alone or cocultured with LSECs. Moreover, CLX abrogated sICAM-1-induced C26 cell proliferation and C26 secretion of promigratory factors for LSECs and HSCs. Interestingly, CLX reduced the protumoral response of HSC, reducing their migratory potential when stimulated with C26 secretomes and impairing their secretion of chemotactic factors for LSECs and C26 cells and proliferative factors for C26 cells. In vivo, CLX abrogated the prometastatic ability of sICAM-1-activated C26 cells while reducing liver metastasis. COX-2 inhibition blocked the creation of a favorable tumor microenvironment (TME) by hindering the intratumoral recruitment of activated HSCs and macrophages in addition to the accumulation of fibrillar collagen. These results point to COX-2 being a key modulator of processes initiated by host ICAM-1 during tumor cell/LSEC/HSC crosstalk, leading to the creation of a prometastatic TME in the liver.
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Project description:Metabolic reprogramming, especially Warburg effect, is a key event in tumor initiation and progression. ZEB1 plays a vital role in metastasis of various cancers. We previously found that ZEB1 was excessively expressed in hepatocellular carcinoma (HCC) and its high expression was closely correlated with metastasis and recurrence of HCC. We want to know whether glycolytic enzymes are regulated by ZEB1 and contribute to carcinogenesis and metastasis of HCC. Methods: To explore whether ZEB1 could enhance glycolysis in HCC, we knocked down ZEB1 by short hairpin RNA (shRNA) in MHCC-97H and HCC-LM3 cells and performed glucose uptake, lactate production, ECAR and OCR assays. To investigate how ZEB1 enhances glycolysis, the protein levels of glycolytic enzymes were detected in the same cell lines using Western blot. The regulatory effect of ZEB1 on PFKM mRNA level was confirmed by RT-qPCR, luciferase report assay and ChIP assay. In order to assess the role of ZEB1-PFKM axis in cell proliferation, cell counting and CCK-8 assays were performed in MHCC-97H and HCC-LM3 cell lines knocked down for ZEB1 and further re-expressed for either ZEB1 or PFKM or not. To explored whether the ZEB1-PFKM axis also functions in HCC cell migration, invasion and metastasis, the same MHCC-97H and HCC-LM3 cell lines were performed for wound healing assays, transwell assays and colony formation assays, meanwhile, MHCC-97H cell lines were performed for orthotopic liver transplantation assays. Finally, the expression of ZEB1 and PFKM were examined in human liver cancer specimens and non-tumorous liver tissues using immunohistochemical and Western blot. Results: We found that ZEB1 transcriptionally upregulates the expression of the muscle isoform of phosphofructokinase-1 (PFKM), a rate-limiting enzyme in glycolysis. Intriguingly, a non-classic ZEB1-binding sequence in the promoter region of PFKM was identified through which ZEB1 directly activates the transcription of PFKM. Silencing of ZEB1 in MHCC-97H and HCC-LM3 cell leads to impaired PFKM expression, glycolysis, proliferation and invasion, and such impairments are rescued by exogenous expression of PFKM. Importantly, in-situ HCC xenograft assays and studies from TCGA database demonstrate that ZEB1-PFKM axis is crucial for carcinogenesis and metastasis of HCC. Conclusions: Our study reveals a novel mechanism of ZEB1 in promoting HCC by activating the transcription of PFKM, establishing the direct link of ZEB1 to the promotion of glycolysis and Warburg effect and suggesting that inhibition of ZEB1 transcriptional activity toward PFKM may be a potential therapeutic strategy for HCC.
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Project description:ObjectiveClinical evidence suggests an association between oxidative stress and vascular disease, and in vitro studies have demonstrated that reactive oxygen species can have prothrombotic effects on vascular and blood cells. It remains unclear, however, whether elevated levels of reactive oxygen species accelerate susceptibility to experimental thrombosis in vivo.Approach and resultsUsing a murine model with genetic deficiency in superoxide dismutase-1 (SOD1), we measured susceptibility to carotid artery thrombosis in response to photochemical injury. We found that SOD1-deficient (Sod1(-/-)) mice formed stable arterial occlusions significantly faster than wild-type (Sod1(+/+)) mice (P<0.05). Sod1(-/-) mice also developed significantly larger venous thrombi than Sod1(+/+) mice after inferior vena cava ligation (P<0.05). Activation of protein C by thrombin in lung was diminished in Sod1(-/-) mice (P<0.05 versus Sod1(+/+) mice), and generation of activated protein C in response to infusion of thrombin in vivo was decreased in Sod1(-/-) mice (P<0.05 versus Sod1(+/+) mice). SOD1 deficiency had no effect on the expression of thrombomodulin, endothelial protein C receptor, or tissue factor in lung or levels of protein C in plasma. Exposure of human thrombomodulin to superoxide in vitro caused oxidation of multiple methionine residues, including critical methionine 388, and a 40% decrease in thrombomodulin-dependent activation of protein C (P<0.05). SOD and catalase protected against superoxide-induced methionine oxidation and restored protein C activation in vitro (P<0.05).ConclusionsSOD prevents thrombomodulin methionine oxidation, promotes protein C activation, and protects against arterial and venous thrombosis in mice.
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