Tspan8 is expressed in breast cancer and regulates E-cadherin/catenin signalling and metastasis accompanied by increased circulating extracellular vesicles.

Tspan8 exhibits a functional role in many cancer types including pancreatic, colorectal, oesophagus carcinoma, and melanoma. We present a first study on the expression and function of Tspan8 in breast cancer. Tspan8 protein was present in the majority of human primary breast cancer lesions and metastases in the brain, bone, lung, and liver. In a syngeneic rat breast cancer model, Tspan8+ tumours formed multiple liver and spleen metastases, while Tspan8- tumours exhibited a significantly diminished ability to metastasise, indicating a role of Tspan8 in metastases. Addressing the underlying molecular mechanisms, we discovered that Tspan8 can mediate up-regulation of E-cadherin and down-regulation of Twist, p120-catenin, and ß-catenin target genes accompanied by the change of cell phenotype, resembling the mesenchymal-epithelial transition. Furthermore, Tspan8+ cells exhibited enhanced cell-cell adhesion, diminished motility, and decreased sensitivity to irradiation. As a regulator of the content and function of extracellular vesicles (EVs), Tspan8 mediated a several-fold increase in EV number in cell culture and the circulation of tumour-bearing animals. We observed increased protein levels of E-cadherin and p120-catenin in these EVs; furthermore, Tspan8 and p120-catenin were co-immunoprecipitated, indicating that they may interact with each other. Altogether, our findings show the presence of Tspan8 in breast cancer primary lesion and metastases and indicate its role as a regulator of cell behaviour and EV release in breast cancer. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Innate extracellular vesicles from melanoma patients suppress ß-catenin in tumor cells by miRNA-34a.

Upon tumor development, new extracellular vesicles appear in circulation. Our knowledge of their relative abundance, function, and overall impact on cancer development is still preliminary. Here, we demonstrate that plasma extracellular vesicles (pEVs) of non-tumor origin are persistently increased in untreated and post-excision melanoma patients, exhibiting strong suppressive effects on the proliferation of tumor cells. Plasma vesicle numbers, miRNAs, and protein levels were elevated two- to tenfold and detected many years after tumor resection. The vesicles revealed individual and clinical stage-specific miRNA profiles as well as active ADAM10. However, whereas pEV from patients preventing tumor relapse down-regulated ß-catenin and blocked tumor cell proliferation in an miR-34a-dependent manner, pEV from metastatic patients lost this ability and stimulated ß-catenin-mediated transcription. Cancer-induced pEV may constitute an innate immune mechanism suppressing tumor cell activity including that of residual cancer cells present after primary surgery.

Multiepitope tissue analysis reveals SPPL3-mediated ADAM10 activation as a key step in the transformation of melanocytes.

The evolution of cancer is characterized by the appearance of specific mutations, but these mutations are translated into proteins that must cooperate to induce malignant transformation. Using a systemic approach with the multiepitope ligand cartography (MELC) technology, we analyzed protein expression profiles (PEPs) in nevi and BRAFV600E-positive superficial spreading melanomas (SSMs) from patient tissues to identify key transformation events. The PEPs in nevi and SSMs differed predominantly in the abundance of specific antigens, but the PEPs of nevi- and melanoma-associated keratinocytes gradually changed during the transformation process. A stepwise change in PEP with similar properties occurred in keratinocytes cocultured with melanoma cells. Analysis of the individual steps indicated that activation of the metalloproteinase ADAM10 by signal peptide peptidase-like 3 (SPPL3) triggered by mutant BRAFV600E was a critical transformation event. SPPL3-mediated ADAM10 activation involved the translocation of SPPL3 and ADAM10 into Rab4- or Rab27-positive endosomal compartments. This endosomal translocation, and hence ADAM10 activation, was inhibited by the presence of the tumor suppressor PTEN. Our findings suggest that systematic tissue antigen analysis could complement whole-genome approaches to provide more insight into cancer development.

HIV-Nef and ADAM17-Containing Plasma Extracellular Vesicles Induce and Correlate with Immune Pathogenesis in Chronic HIV Infection.

Antiretroviral therapy (ART) efficiently suppresses HIV replication but immune activation and low CD4 T cell counts often persist. The underlying mechanism of this ART-resistant pathogenesis is not clear. We observed that levels of plasma extracellular vesicles (pEV) are strongly elevated in HIV infection and do not decline during ART. Surprisingly, these vesicles contained the viral accessory proteins Nef and Vpu, which are assumed to be not expressed under efficient ART, as well as pro-inflammatory effectors, including activated ADAM17. HIV pEV were characterized by the presence of activated αvß3 and absence of CD81 and Tsg101. Correlating with immune activation, peripheral monocytes ingested large amounts of pEV, giving rise to an increased population of CD1c(+) CD14(+) cells that secreted inflammatory cytokines. Importantly, the pro-inflammatory content, particularly ADAM17 activity, correlated with low T cell counts. Preliminary evidence suggested that HIV pEV derived from peripheral mononuclear cells and from an unknown myeloid cell population. In summary we propose an important role of pro-inflammatory pEV in chronic HIV infection due to ongoing viral Nef activity.

HIV Nef, paxillin, and Pak1/2 regulate activation and secretion of TACE/ADAM10 proteases.

The HIV Nef protein recruits the polycomb protein Eed and mimics an integrin receptor signal for reasons that are not entirely clear. Here we demonstrate that Nef and Eed complex with the integrin effector paxillin to recruit and activate TNFα converting enzyme (TACE alias ADAM 17) and its close relative ADAM10. The activated proteases cleaved proTNFα and were shuttled into extracellular vesicles (EVs). Peripheral blood mononuclear cells that ingested these EVs released TNFα. Analyzing the mechanism, we found that Pak2, an established host cell effector of Nef, phosphorylated paxillin on Ser272/274 to induce TACE-paxillin association and shuttling into EVs via lipid rafts. Conversely, Pak1 phosphorylated paxillin on Ser258, which inhibited TACE association and lipid raft transfer. Interestingly, melanoma cells used an identical mechanism to shuttle predominantly ADAM10 into EVs. We conclude that HIV-1 and cancer cells exploit a paxillin/integrin-controlled mechanism to release TACE/ADAM10-containing vesicles, ensuring better proliferation/growth conditions in their microenvironment.