TM9SF4 is a novel V-ATPase-interacting protein that modulates tumor pH alterations associated with drug resistance and invasiveness of colon cancer cells

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Cannibalism of Live Lymphocytes by Human Metastatic but Not Primary Melanoma Cells

The phenomenon of cell cannibalism, which generally refers to the engulfment of cells within other cells, was described in malignant tumors, but its biological significance is still largely unknown. In the present study, we investigated the occurrence, the in vivo relevance, and the underlying mechanisms of cannibalism in human melanoma. As first evidence, we observed that tumor cannibalism was clearly detectable in vivo in metastatic lesions of melanoma and often involved T cells, which could be found in a degraded state within tumor cells. Then, in vitro experiments confirmed that cannibalism of T cells was a property of metastatic melanoma cells but not of primary melanoma cells. In particular, morphologic analyses, including time-lapse cinematography and electron microscopy, revealed a sequence of events, in which metastatic melanoma cells were able to engulf and digest live autologous melanoma-specific CD8+ T cells. Importantly, this cannibalistic activity significantly increased metastatic melanoma cell survival, particularly under starvation condition, supporting the evidence that tumor cells may use the eating of live lymphocytes as a way to ‘‘feed’’ in condition of low nutrient supply. The mechanism underlying cannibalism involved a complex framework, including lysosomal protease cathepsin B activity, caveolae formation, and ezrin cytoskeleton integrity and function. In conclusion, our study shows that human metastatic melanoma cells may eat live T cells, which are instead programmed to kill them, suggesting a novel mechanism of tumor immune escape. Moreover, our data suggest that cannibalism may represent a sort of ‘‘feeding’’ activity aimed at sustaining survival and progression of malignant tumor cells in an unfavorable microenvironment. (Cancer Res 2006; 66(7): 3629-38

High Levels of Exosomes Expressing CD63 and Caveolin-1 in Plasma of Melanoma Patients

BACKGROUND: Metastatic melanoma is an untreatable cancer lacking reliable and non-invasive markers of disease progression. Exosomes are small vesicles secreted by normal as well as tumor cells. Human tumor-derived exosomes are involved in malignant progression and we evaluated the presence of exosomes in plasma of melanoma patients as a potential tool for cancer screening and follow-up. METHODOLOGY/PRINCIPAL FINDINGS: We designed an in-house sandwich ELISA (Exotest) to capture and quantify exosomes in plasma based on expression of housekeeping proteins (CD63 and Rab-5b) and a tumor-associated marker (caveolin-1). Western blot and flow cytometry analysis of exosomes were used to confirm the Exotest-based findings. The Exotest allowed sensitive detection and quantification of exosomes purified from human tumor cell culture supernatants and plasma from SCID mice engrafted with human melanoma. Plasma levels of exosomes in melanoma-engrafted SCID mice correlated to tumor size. We evaluated the levels of plasma exosomes expressing CD63 and caveolin-1 in melanoma patients (n = 90) and healthy donors (n = 58). Consistently, plasma exosomes expressing CD63 (504+/-315) or caveolin-1 (619+/-310) were significantly increased in melanoma patients as compared to healthy donors (223+/-125 and 228+/-102, respectively). While the Exotest for CD63+ plasma exosomes had limited sensitivity (43%) the Exotest for detection of caveolin-1+ plasma exosomes showed a higher sensitivity (68%). Moreover, caveolin-1+ plasma exosomes were significantly increased with respect to CD63+ exosomes in the patients group. CONCLUSIONS/SIGNIFICANCE: We describe a new non-invasive assay allowing detection and quantification of human exosomes in plasma of melanoma patients. Our results suggest that the Exotest for detection of plasma exosomes carrying tumor-associated antigens may represent a novel tool for clinical management of cancer patients

Identification and relevance of the CD95-binding domain in the N-terminal region of ezrin

The CD95 (Fas/APO-1) linkage to the actin cytoskeleton through ezrin is an essential requirement for susceptibility to the CD95-mediated apoptosis in CD4+ T cells. We have previously shown that moesin was not involved in the binding to CD95. Here we further support the specificity of the ezrin/CD95 binding, showing that radixin did not bind CD95. The ezrin region specifically and directly involved in the binding to CD95 was located in the middle lobe of the ezrin FERM domain, between amino acids 149 and 168. In this region, ezrin, radixin, and moesin show 60-65% identity, as compared with the 86% identity in the whole FERM domain. Transfection of two different human cell lines with a green fluorescent protein-tagged ezrin mutated in the CD95-binding epitope, induced a marked inhibition of CD95-mediated apoptosis. In these cells, the mutated ezrin did not co-localize or co-immunoprecipitate with CD95. Further analysis showed that the mutated ezrin, while unable to bind CD95, was fully able to bind actin, thus preventing the actin linkage to CD95. Altogether, our results support the specificity of ezrin in the association to CD95 and the importance of the ezrin-to-CD95 linkage in CD95-mediated apoptosis. Moreover, this study suggests that a major role of ezrin is to connect CD95 to actin, thus allowing the CD95 polarization on the cells and the occurrence of the following multiple cascades of the CD95 pathway

Morphological analysis of the pancreatic beta cells of diabetic female rats at different ages of life

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Emerging insights on the role of V-ATPase in human diseases: Therapeutic challenges and opportunities

The control of the intracellular pH is vital for the survival of all organisms. Membrane transporters, both at the plasma and intracellular membranes, are key players in maintaining a finely tuned pH balance between intra- and extracellular spaces, and therefore in cellular homeostasis. V-ATPase is a housekeeping ATP-driven proton pump highly conserved among prokaryotes and eukaryotes. This proton pump, which exhibits a complex multisubunit structure based on cell type-specific isoforms, is essential for pH regulation and for a multitude of ubiquitous and specialized functions. Thus, it is not surprising that V-ATPase aberrant overexpression, mislocalization, and mutations in V-ATPase subunit-encoding genes have been associated with several human diseases. However, the ubiquitous expression of this transporter and the high toxicity driven by its off-target inhibition, renders V-ATPase-directed therapies very challenging and increases the need for selective strategies. Here we review emerging evidence linking V-ATPase and both inherited and acquired human diseases, explore the therapeutic challenges and opportunities envisaged from recent data, and advance future research avenues. We highlight the importance of V-ATPases with unique subunit isoform molecular signatures and disease-associated isoforms to design selective V-ATPase-directed therapies. We also discuss the rational design of drug development pipelines and cutting-edge methodological approaches toward V-ATPase-centered drug discovery. Diseases like cancer, osteoporosis, and even fungal infections can benefit from V-ATPase-directed therapies.European Regional Development Fund, Grant/Award Number: BioTecNorte operation (NORTE‐01‐0145‐FEDER‐000004); Fundação para a Ciência e a Tecnologia, Grant/Award Numbers: PhD fellowship (PD/BD/128032/2016), Strategic funding of UIDB/04469/2020 unit and of “Contrato‐Programa” UIDB/04050/2020info:eu-repo/semantics/publishedVersio

The human homologue of Dictyostelium discoideum phg1A is expressed by human metastatic melanoma cells

Tumour cannibalism is a characteristic of malignancy and metastatic behaviour. This atypical phagocytic activity is a crucial survival option for tumours in conditions of low nutrient supply, and has some similarities to the phagocytic activity of unicellular microorganisms. In fact, Dictyostelium discoideum has been used widely as a model to study phagocytosis. Recently, phg1A has been described as a protein that is primarily involved in the phagocytic process of this microorganism. The closest human homologue to phg1A is transmembrane 9 superfamily protein member 4 (TM9SF4). Here, we report that TM9SF4 is highly expressed in human malignant melanoma cells deriving from metastatic lesions, whereas it is undetectable in healthy human tissues and cells. TM9SF4 is predominantly expressed in acidic vesicles of melanoma cells, in which it co-localizes with the early endosome antigens Rab5 and early endosome antigen 1. TM9SF4 silencing induced marked inhibition of cannibal activity, which is consistent with a derangement of intracellular pH gradients, with alkalinization of acidic vesicles and acidification of the cell cytosol. We propose TM9SF4 as a new marker of malignancy, representing a potential new target for anti-tumour strategies with a specific role in tumour cannibalism and in the establishment of a metastatic phenotype