Triazole derivatives inhibit the VOR complex-mediated nuclear transport of extracellular particles: Potential application in cancer and HIV-1 infection.

Extracellular vesicles (EVs) appear to play an important role in intercellular communication in various physiological processes and pathological conditions such as cancer. Like enveloped viruses, EVs can transport their contents into the nucleus of recipient cells, and a new intracellular pathway has been described to explain the nuclear shuttling of EV cargoes. It involves a tripartite protein complex consisting of vesicle-associated membrane protein-associated protein A (VAP-A), oxysterol-binding protein (OSBP)-related protein-3 (ORP3) and late endosome-associated Rab7 allowing late endosome entry into the nucleoplasmic reticulum. Rab7 binding to ORP3-VAP-A complex can be blocked by the FDA-approved antifungal drug itraconazole. Here, we design a new series of smaller triazole derivatives, which lack the dioxolane moiety responsible for the antifungal function, acting on the hydrophobic sterol-binding pocket of ORP3 and evaluate their structure-activity relationship through inhibition of VOR interactions and nuclear transfer of EV and HIV-1 cargoes. Our investigation reveals that the most effective compounds that prevent nuclear transfer of EV cargo and productive infection by VSV-G-pseudotyped HIV-1 are those with a side chain between 1 and 4 carbons, linear or branched (methyl) on the triazolone region. These potent chemical drugs could find clinical applications either for nuclear transfer of cancer-derived EVs that impact metastasis or viral infection.

The F-actin bundler SWAP-70 promotes tumor metastasis.

Dynamic rearrangements of the F-actin cytoskeleton are a hallmark of tumor metastasis. Thus, proteins that govern F-actin rearrangements are of major interest for understanding metastasis and potential therapies. We hypothesized that the unique F-actin binding and bundling protein SWAP-70 contributes importantly to metastasis. Orthotopic, ectopic, and short-term tail vein injection mouse breast and lung cancer models revealed a strong positive dependence of lung and bone metastasis on SWAP-70. Breast cancer cell growth, migration, adhesion, and invasion assays revealed SWAP-70's key role in these metastasis-related cell features and the requirement for SWAP-70 to bind F-actin. Biophysical experiments showed that tumor cell stiffness and deformability are negatively modulated by SWAP-70. Together, we present a hitherto undescribed, unique F-actin modulator as an important contributor to tumor metastasis.

Extracellular lipidosomes containing lipid droplets and mitochondria are released during melanoma cell division.

BACKGROUND: The incidence of melanoma is increasing worldwide. Since metastatic melanoma is highly aggressive, it is important to decipher all the biological aspects of melanoma cells. In this context, we have previously shown that metastatic FEMX-I melanoma cells release small (< 150 nm) extracellular vesicles (EVs) known as exosomes and ectosomes containing the stem (and cancer stem) cell antigenic marker CD133. EVs play an important role in intercellular communication, which could have a micro-environmental impact on surrounding tissues. RESULTS: We report here a new type of large CD133+ EVs released by FEMX-I cells. Their sizes range from 2 to 6 µm and they contain lipid droplets and mitochondria. Real-time video microscopy revealed that these EVs originate from the lipid droplet-enriched cell extremities that did not completely retract during the cell division process. Once released, they can be taken up by other cells. Silencing CD133 significantly affected the cellular distribution of lipid droplets, with a re-localization around the nuclear compartment. As a result, the formation of large EVs containing lipid droplets was severely compromised. CONCLUSION: Given the biochemical effect of lipid droplets and mitochondria and/or their complexes on cell metabolism, the release and uptake of these new large CD133+ EVs from dividing aggressive melanoma cells can influence both donor and recipient cells, and therefore impact melanoma growth and dissemination.

HIV-1-induced nuclear invaginations mediated by VAP-A, ORP3, and Rab7 complex explain infection of activated T cells.

The mechanism of human immunodeficiency virus 1 (HIV-1) nuclear entry, required for productive infection, is not fully understood. Here, we report that in HeLa cells and activated CD4+ T cells infected with HIV-1 pseudotyped with VSV-G and native Env protein, respectively, Rab7+ late endosomes containing endocytosed HIV-1 promote the formation of nuclear envelope invaginations (NEIs) by a molecular mechanism involving the VOR complex, composed of the outer nuclear membrane protein VAP-A, hyperphosphorylated ORP3 and Rab7. Silencing VAP-A or ORP3 and drug-mediated impairment of Rab7 binding to ORP3-VAP-A inhibited the nuclear transfer of the HIV-1 components and productive infection. In HIV-1-resistant quiescent CD4+ T cells, ORP3 was not hyperphosphorylated and neither VOR complex nor NEIs were formed. This new cellular pathway and its molecular players are potential therapeutic targets, perhaps shared by other viruses that require nuclear entry to complete their life cycle.

Mesenchymal stromal cell-associated migrasomes: a new source of chemoattractant for cells of hematopoietic origin.

BACKGROUND: Multipotent mesenchymal stromal cells (MSCs) are precursors of various cell types. Through soluble factors, direct cell-cell interactions and other intercellular communication mechanisms such as extracellular vesicles and tunneling nanotubes, MSCs support tissue homeostasis. In the bone marrow microenvironment, they promote hematopoiesis. The interaction between MSCs and cancer cells enhances the cancer and metastatic potential. Here, we have demonstrated that plastic-adherent MSCs isolated from human bone marrow generate migrasomes, a newly discovered organelle playing a role in intercellular communication. RESULTS: Migrasomes are forming a network with retraction fibers behind the migrating MSCs or surrounding them after membrane retraction. The MSC markers, CD44, CD73, CD90, CD105 and CD166 are present on the migrasome network, the latter being specific to migrasomes. Some migrasomes harbor the late endosomal GTPase Rab7 and exosomal marker CD63 indicating the presence of multivesicular bodies. Stromal cell-derived factor 1 (SDF-1) was detected in migrasomes, suggesting that they play a chemoattractant role. Co-cultures with KG-1a leukemic cells or primary CD34+ hematopoietic progenitors revealed that MSC-associated migrasomes attracted them, a process intercepted by the addition of AMD3100, a specific CXCR4 receptor inhibitor, or recombinant SDF-1. An antibody directed against CD166 reduced the association of hematopoietic cells and MSC-associated migrasomes. In contrast to primary CD34+ progenitors, leukemic cells can take up migrasomes. CONCLUSION: Overall, we described a novel mechanism used by MSCs to communicate with cells of hematopoietic origin and further studies are needed to decipher all biological aspects of migrasomes in the healthy and transformed bone marrow microenvironment. Video Abstract.

Anti-Human CD9 Fab Fragment Antibody Blocks the Extracellular Vesicle-Mediated Increase in Malignancy of Colon Cancer Cells.

Intercellular communication between cancer cells themselves or with healthy cells in the tumor microenvironment and/or pre-metastatic sites plays an important role in cancer progression and metastasis. In addition to ligand-receptor signaling complexes, extracellular vesicles (EVs) are emerging as novel mediators of intercellular communication both in tissue homeostasis and in diseases such as cancer. EV-mediated transfer of molecular activities impacting morphological features and cell motility from highly metastatic SW620 cells to non-metastatic SW480 cells is a good in vitro example to illustrate the increased malignancy of colorectal cancer leading to its transformation and aggressive behavior. In an attempt to intercept the intercellular communication promoted by EVs, we recently developed a monovalent Fab fragment antibody directed against human CD9 tetraspanin and showed its effectiveness in blocking the internalization of melanoma cell-derived EVs and the nuclear transfer of their cargo proteins into recipient cells. Here, we employed the SW480/SW620 model to investigate the anti-cancer potential of the anti-CD9 Fab antibody. We first demonstrated that most EVs derived from SW620 cells contain CD9, making them potential targets. We then found that the anti-CD9 Fab antibody, but not the corresponding divalent antibody, prevented internalization of EVs from SW620 cells into SW480 cells, thereby inhibiting their phenotypic transformation, i.e., the change from a mesenchymal-like morphology to a rounded amoeboid-like shape with membrane blebbing, and thus preventing increased cell migration. Intercepting EV-mediated intercellular communication in the tumor niche with an anti-CD9 Fab antibody, combined with direct targeting of cancer cells, could lead to the development of new anti-cancer therapeutic strategies.

Decoding Single Cell Morphology in Osteotropic Breast Cancer Cells for Dissecting Their Migratory, Molecular and Biophysical Heterogeneity.

Breast cancer is a heterogeneous disease and the mechanistic framework for differential osteotropism among intrinsic breast cancer subtypes is unknown. Hypothesizing that cell morphology could be an integrated readout for the functional state of a cancer cell, we established a catalogue of the migratory, molecular and biophysical traits of MDA-MB-231 breast cancer cells, compared it with two enhanced bone-seeking derivative cell lines and integrated these findings with single cell morphology profiles. Such knowledge could be essential for predicting metastatic capacities in breast cancer. High-resolution microscopy revealed a heterogeneous and specific spectrum of single cell morphologies in bone-seeking cells, which correlated with differential migration and stiffness. While parental MDA-MB-231 cells showed long and dynamic membrane protrusions and were enriched in motile cells with continuous and mesenchymal cell migration, bone-seeking cells appeared with discontinuous mesenchymal or amoeboid-like migration. Although non-responsive to CXCL12, bone-seeking cells responded to epidermal growth factor with a morphotype shift and differential expression of genes controlling cell shape and directional migration. Hence, single cell morphology encodes the molecular, migratory and biophysical architecture of breast cancer cells and is specifically altered among osteotropic phenotypes. Quantitative morpho-profiling could aid in dissecting breast cancer heterogeneity and in refining clinically relevant intrinsic breast cancer subtypes.

Itraconazole inhibits nuclear delivery of extracellular vesicle cargo by disrupting the entry of late endosomes into the nucleoplasmic reticulum.

Extracellular vesicles (EVs) are mediators of intercellular communication under both healthy and pathological conditions, including the induction of pro-metastatic traits, but it is not yet known how and where functional cargoes of EVs are delivered to their targets in host cell compartments. We have described that after endocytosis, EVs reach Rab7+ late endosomes and a fraction of these enter the nucleoplasmic reticulum and transport EV biomaterials to the host cell nucleoplasm. Their entry therein and docking to outer nuclear membrane occur through a tripartite complex formed by the proteins VAP-A, ORP3 and Rab7 (VOR complex). Here, we report that the antifungal compound itraconazole (ICZ), but not its main metabolite hydroxy-ICZ or ketoconazole, disrupts the binding of Rab7 to ORP3-VAP-A complexes, leading to inhibition of EV-mediated pro-metastatic morphological changes including cell migration behaviour of colon cancer cells. With novel, smaller chemical drugs, inhibition of the VOR complex was maintained, although the ICZ moieties responsible for antifungal activity and interference with intracellular cholesterol distribution were removed. Knowing that cancer cells hijack their microenvironment and that EVs derived from them determine the pre-metastatic niche, small-sized inhibitors of nuclear transfer of EV cargo into host cells could find cancer therapeutic applications, particularly in combination with direct targeting of cancer cells.

CD9, a tetraspanin target for cancer therapy?

In the present minireview, we intend to provide a brief history of the field of CD9 involvement in oncogenesis and in the metastatic process of cancer, considering its potential value as a tumor-associated antigenic target. Over the years, CD9 has been identified as a favorable prognostic marker or predictor of metastatic potential depending on the cancer type. To understand its implications in cancer beside its use as an antigenic biomarker, it is essential to know its physiological functions, including its molecular partners in a given cell system. Moreover, the discovery that CD9 is one of the most specific and broadly expressed markers of extracellular membrane vesicles, nanometer-sized entities that are released into extracellular space and various physiological body fluids and play a role in intercellular communication under physiological and pathological conditions, notably the establishment of cancer metastases, has added a new dimension to our knowledge of CD9 function in cancer. Here, we will discuss these issues as well as the possible cancer therapeutic implications of CD9, their limitations, and pitfalls.

Uptake and Fate of Extracellular Membrane Vesicles: Nucleoplasmic Reticulum-Associated Late Endosomes as a New Gate to Intercellular Communication.

Extracellular membrane vesicles (EVs) are emerging as new vehicles in intercellular communication, but how the biological information contained in EVs is shared between cells remains elusive. Several mechanisms have been described to explain their release from donor cells and the initial step of their uptake by recipient cells, which triggers a cellular response. Yet, the intracellular routes and subcellular fate of EV content upon internalization remain poorly characterized. This is particularly true for EV-associated proteins and nucleic acids that shuttle to the nucleus of host cells. In this review, we will describe and discuss the release of EVs from donor cells, their uptake by recipient cells, and the fate of their cargoes, focusing on a novel intracellular route wherein small GTPase Rab7+ late endosomes containing endocytosed EVs enter into nuclear envelope invaginations and deliver their cargo components to the nucleoplasm of recipient cells. A tripartite protein complex composed of (VAMP)-associated protein A (VAP-A), oxysterol-binding protein (OSBP)-related protein-3 (ORP3), and Rab7 is essential for the transfer of EV-derived components to the nuclear compartment by orchestrating the particular localization of late endosomes in the nucleoplasmic reticulum.

Prominins control ciliary length throughout the animal kingdom: New lessons from human prominin-1 and zebrafish prominin-3.

Prominins (proms) are transmembrane glycoproteins conserved throughout the animal kingdom. They are associated with plasma membrane protrusions, such as primary cilia, as well as extracellular vesicles derived thereof. Primary cilia host numerous signaling pathways affected in diseases known as ciliopathies. Human PROM1 (CD133) is detected in both somatic and cancer stem cells and is also expressed in terminally differentiated epithelial and photoreceptor cells. Genetic mutations in the PROM1 gene result in retinal degeneration by impairing the proper formation of the outer segment of photoreceptors, a modified cilium. Here, we investigated the impact of proms on two distinct examples of ciliogenesis. First, we demonstrate that the overexpression of a dominant-negative mutant variant of human PROM1 (i.e. mutation Y819F/Y828F) significantly decreases ciliary length in Madin-Darby canine kidney cells. These results contrast strongly to the previously observed enhancing effect of WT PROM1 on ciliary length. Mechanistically, the mutation impeded the interaction of PROM1 with ADP-ribosylation factor-like protein 13B, a key regulator of ciliary length. Second, we observed that in vivo knockdown of prom3 in zebrafish alters the number and length of monocilia in the Kupffer's vesicle, resulting in molecular and anatomical defects in the left-right asymmetry. These distinct loss-of-function approaches in two biological systems reveal that prom proteins are critical for the integrity and function of cilia. Our data provide new insights into ciliogenesis and might be of particular interest for investigations of the etiologies of ciliopathies.

ALCAM contributes to brain metastasis formation in non-small-cell lung cancer through interaction with the vascular endothelium.

BACKGROUND: Brain metastasis (BM) in non-small-cell lung cancer (NSCLC) has a very poor prognosis. Recent studies have demonstrated the importance of cell adhesion molecules in tumor metastasis. The aim of our study was to investigate the role of activated leukocyte cell adhesion molecule (ALCAM) in BM formation in NSCLC. METHODS: Immunohistochemical analysis was performed on 143 NSCLC primary tumors and BM. A correlation between clinicopathological parameters and survival was developed. Biological properties of ALCAM were assessed in vitro by gene ablation using CRISPR/Cas9 technology in the NCI-H460 NSCLC cell line and in vivo by intracranial and intracardial cell injection of NCI-H460 cells in NMRI-Foxn1nu/nu mice. RESULTS: ALCAM expression was significantly upregulated in NSCLC brain metastasis (P = 0.023) with a de novo expression of ALCAM in 31.2% of BM. Moderate/strong ALCAM expression in both primary NSCLC and brain metastasis was associated with shortened survival. Functional analysis of an ALCAM knock-out (KO) cell line showed a significantly decreased cell adhesion capacity to human brain endothelial cells by 38% (P = 0.045). In vivo studies showed significantly lower tumor cell dissemination in mice injected with ALCAM-KO cells in both mouse models, and both the number and size of BM were significantly diminished in ALCAM depleted tumors. CONCLUSIONS: Our findings suggest that elevated levels of ALCAM expression promote BM formation in NSCLC through increased tumor cell dissemination and interaction with the brain endothelial cells. Therefore, ALCAM could be targeted to reduce the occurrence of BM. KEY POINTS: 1. ALCAM expression associates with poor prognosis and brain metastasis in NSCLC.2. ALCAM mediates interaction of NSCLC tumor cells with brain vascular endothelium.3. ALCAM might represent a novel preventive target to reduce the occurrence of BM in NSCLC.

Transit amplifying cells coordinate mouse incisor mesenchymal stem cell activation.

Stem cells (SCs) receive inductive cues from the surrounding microenvironment and cells. Limited molecular evidence has connected tissue-specific mesenchymal stem cells (MSCs) with mesenchymal transit amplifying cells (MTACs). Using mouse incisor as the model, we discover a population of MSCs neibouring to the MTACs and epithelial SCs. With Notch signaling as the key regulator, we disclose molecular proof and lineage tracing evidence showing the distinct MSCs contribute to incisor MTACs and the other mesenchymal cell lineages. MTACs can feedback and regulate the homeostasis and activation of CL-MSCs through Delta-like 1 homolog (Dlk1), which balances MSCs-MTACs number and the lineage differentiation. Dlk1's function on SCs priming and self-renewal depends on its biological forms and its gene expression is under dynamic epigenetic control. Our findings can be validated in clinical samples and applied to accelerate tooth wound healing, providing an intriguing insight of how to direct SCs towards tissue regeneration.

Anti-human CD9 antibody Fab fragment impairs the internalization of extracellular vesicles and the nuclear transfer of their cargo proteins.

The intercellular communication mediated by extracellular vesicles (EVs) has gained international interest during the last decade. Interfering with the mechanisms regulating this cellular process might find application particularly in oncology where cancer cell-derived EVs play a role in tumour microenvironment transformation. Although several mechanisms were ascribed to explain the internalization of EVs, little is our knowledge about the fate of their cargos, which are crucial to mediate their function. We recently demonstrated a new intracellular pathway in which a fraction of endocytosed EV-associated proteins is transported into the nucleoplasm of the host cell via a subpopulation of late endosomes penetrating into the nucleoplasmic reticulum. Silencing tetraspanin CD9 both in EVs and recipient cells strongly decreased the endocytosis of EVs and abolished the nuclear transfer of their cargos. Here, we investigated whether monovalent Fab fragments derived from 5H9 anti-CD9 monoclonal antibody (referred hereafter as CD9 Fab) interfered with these cellular processes. To monitor the intracellular transport of proteins, we used fluorescent EVs containing CD9-green fluorescent protein fusion protein and various melanoma cell lines and bone marrow-derived mesenchymal stromal cells as recipient cells. Interestingly, CD9 Fab considerably reduced EV uptake and the nuclear transfer of their proteins in all examined cells. In contrast, the divalent CD9 antibody stimulated both events. By impeding intercellular communication in the tumour microenvironment, CD9 Fab-mediated inhibition of EV uptake, combined with direct targeting of cancerous cells could lead to the development of novel anti-melanoma therapeutic strategies.

Prominin-1 (CD133) modulates the architecture and dynamics of microvilli.

Prominin-1 is a cell surface biomarker that allows the identification of stem and cancer stem cells from different organs. It is also expressed in several differentiated epithelial and non-epithelial cells. Irrespective of the cell type, prominin-1 is associated with plasma membrane protrusions. Here, we investigate its impact on the architecture of membrane protrusions using microvilli of Madin-Darby canine kidney cells as the main model. Our high-resolution analysis revealed that upon the overexpression of prominin-1 the number of microvilli and clusters of them increased. Microvilli with branched and/or knob-like morphologies were observed and stimulated by mutations in the ganglioside-binding site of prominin-1. The altered phenotypes were caused by the interaction of prominin-1 with phosphoinositide 3-kinase and Arp2/3 complex. Mutation of tyrosine 828 of prominin-1 impaired its phosphorylation and thereby inhibited the aforementioned interactions abolishing altered microvilli. This suggests that the interplay of prominin-1-ganglioside membrane complexes, phosphoinositide 3-kinase and cytoskeleton components regulates microvillar architecture. Lastly, the expression of prominin-1 and its mutants modified the structure of filopodia emerging from fibroblast-like cells and silencing human prominin-1 in primary hematopoietic stem cells resulted in the loss of uropod-associated microvilli. Altogether, these findings strengthen the role of prominin-1 as an organizer of cellular protrusions.

Prominin-1 controls stem cell activation by orchestrating ciliary dynamics.

Proper temporal and spatial activation of stem cells relies on highly coordinated cell signaling. The primary cilium is the sensory organelle that is responsible for transmitting extracellular signals into a cell. Primary cilium size, architecture, and assembly-disassembly dynamics are under rigid cell cycle-dependent control. Using mouse incisor tooth epithelia as a model, we show that ciliary dynamics in stem cells require the proper functions of a cholesterol-binding membrane glycoprotein, Prominin-1 (Prom1/CD133), which controls sequential recruitment of ciliary membrane components, histone deacetylase, and transcription factors. Nuclear translocation of Prom1 and these molecules is particularly evident in transit amplifying cells, the immediate derivatives of stem cells. The absence of Prom1 impairs ciliary dynamics and abolishes the growth stimulation effects of sonic hedgehog (SHH) treatment, resulting in the disruption of stem cell quiescence maintenance and activation. We propose that Prom1 is a key regulator ensuring appropriate response of stem cells to extracellular signals, with important implications for development, regeneration, and diseases.

VAMP-associated protein-A and oxysterol-binding protein-related protein 3 promote the entry of late endosomes into the nucleoplasmic reticulum.

The endocytic pathway plays an instrumental role in recycling internalized molecules back to the plasma membrane or in directing them to lysosomes for degradation. We recently reported a new role of endosomes-the delivery of components from extracellular vesicles (EVs) to the nucleoplasm of recipient cells. Using indirect immunofluorescence, FRET, immunoisolation techniques, and RNAi, we report here a tripartite protein complex (referred to as the VOR complex) that is essential for the nuclear transfer of EV-derived components by orchestrating the specific localization of late endosomes into nucleoplasmic reticulum. We found that the VOR complex contains the endoplasmic reticulum-localized vesicle-associated membrane protein (VAMP)-associated protein A (VAP-A), the cytoplasmic oxysterol-binding protein-related protein 3 (ORP3), and late endosome-associated small GTPase Rab7. The silencing of VAP-A or ORP3 abrogated the association of Rab7-positive late endosomes with nuclear envelope invaginations and, hence, the transport of endocytosed EV-derived components to the nucleoplasm of recipient cells. We conclude that the VOR complex can be targeted to inhibit EV-mediated intercellular communication, which can have therapeutic potential for managing cancer in which the release of EVs is dysregulated.

SOX2-silenced squamous cell carcinoma: a highly malignant form of esophageal cancer with SOX2 promoter hypermethylation.

This study originally aimed to investigate whether the overexpression of SOX2 is associated with the poor prognosis of patients with squamous cell carcinoma of the esophagus. However, we unexpectedly found that esophageal squamous cell carcinomas completely lacking SOX2 expression showed distinct pathologic features and highly aggressive clinical courses. The study cohort consisted of 113 consecutive patients with esophageal squamous cell carcinoma who underwent surgical resection without neoadjuvant therapy. Immunostaining on tissue microarrays and whole sections revealed that 8/113 (7%) cases were entirely negative for this transcriptional factor. SOX2-negative cancers were histologically less differentiated (P=0.002) and showed higher pT and pStages (P=0.003 and 0.007, respectively) than SOX2-positive cases. A remarkable finding was widespread lymphatic infiltration distant from the primary invasive focus, which was observed in 4 SOX2-negative cancers (50%), but none of the SOX2-positive cases. All separate dysplastic lesions observed in SOX2-negative cases were also SOX2-negative. The negative expression of SOX2 appeared to be an independent poor prognostic factor (OR=7.05, 95% CI=1.27-39.0). No mutations were identified in the coding or non-coding regions of SOX2. Fluorescent in situ hybridization did not show any copy-number variations in this gene. Since the SOX2 promoter contains an extensive CpG island, SOX2-negative cases underwent methylation-specific PCR, which disclosed promoter hypermethylation in all cases. In conclusion, SOX2-silenced squamous cell carcinomas of the esophagus appear to be a minor, but distinct form of malignancy characterized by extensive lymphatic invasion, a poor prognosis, and potential association with multiple SOX2-negative neoplastic lesions. The hypermethylation of the promoter region is seemingly a critical epigenetic event leading to SOX2 silencing.