Francesco Lozupone

Induction of Lymphocyte Apoptosis by Tumor Cell Secretion of FasL-bearing Microvesicles

The hypothesis that FasL expression by tumor cells may impair the in vivo efficacy of antitumor immune responses, through a mechanism known as ‘Fas tumor counterattack,’ has been recently questioned, becoming the object of an intense debate based on conflicting results. Here we definitely show that FasL is indeed detectable in the cytoplasm of melanoma cells and its expression is confined to multivesicular bodies that contain melanosomes. In these structures FasL colocalizes with both melanosomal (i.e., gp100) and lysosomal (i.e., CD63) antigens. Isolated melanosomes express FasL, as detected by Western blot and cytofluorimetry, and they can exert Fas-mediated apoptosis in Jurkat cells. We additionally show that melanosome-containing multivesicular bodies degranulate extracellularly and release FasL-bearing microvesicles, that coexpress both gp100 and CD63 and retain their functional activity in triggering Fas-dependent apoptosis of lymphoid cells. Hence our data provide evidence for a novel mechanism potentially operating in Fas tumor counterattack through the secretion of subcellular particles expressing functional FasL. Such vesicles may form a sort of front line hindering lymphocytes and other immunocompetent cells from entering neoplastic lesions and exert their antitumor activity.

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.

CD95 (APO‐1/Fas) linkage to the actin cytoskeleton through ezrin in human T lymphocytes: a novel regulatory mechanism of the CD95 apoptotic pathway

CD95 (APO‐1/Fas) is a member of the tumor necrosis factor receptor family, which can trigger apoptosis in a variety of cell types. However, little is known of the mechanisms underlying cell susceptibility to CD95‐mediated apoptosis. Here we show that human T cells that are susceptible to CD95‐mediated apoptosis, exhibit a constitutive polarized morphology, and that CD95 colocalizes with ezrin at the site of cellular polarization. In fact, CD95 co‐immunoprecipitates with ezrin exclusively in lymphoblastoid CD4+ T cells and primary long‐term activated T lymphocytes, which are prone to CD95‐mediated apoptosis, but not in short‐term activated T lymphocytes, which are refractory to the same stimuli, even expressing equal levels of CD95 on the cell membrane. Pre‐treatment with ezrin antisense oligonucleotides specifically protected from the CD95‐mediated apoptosis. Moreover, we show that the actin cytoskeleton integrity is essential for this function. These findings strongly suggest that the CD95 cell membrane polarization, through an ezrin‐mediated association with the actin cytoskeleton, is a key intracellular mechanism in rendering human T lymphocytes susceptible to the CD95‐mediated apoptosis.

pH-dependent antitumor activity of proton pump inhibitors against human melanoma is mediated by inhibition of tumor acidity

Metastatic melanoma is associated with poor prognosis and still limited therapeutic options. An innovative treatment approach for this disease is represented by targeting acidosis, a feature characterizing tumor microenvironment and playing an important role in cancer malignancy. Proton pump inhibitors (PPI), such as esomeprazole (ESOM) are prodrugs functionally activated by acidic environment, fostering pH neutralization by inhibiting proton extrusion. We used human melanoma cell lines and xeno‐transplated SCID mice to provide preclinical evidence of ESOM antineoplastic activity. Human melanoma cell lines, characterized by different mutation and signaling profiles, were treated with ESOM in different pH conditions and evaluated for proliferation, viability and cell death. SCID mice engrafted with human melanoma were used to study ESOM administration effects on tumor growth and tumor pH by magnetic resonance spectroscopy (MRS). ESOM inhibited proliferation of melanoma cells in vitro and induced a cytotoxicity strongly boosted by low pH culture conditions. ESOM‐induced tumor cell death occurred via rapid intracellular acidification and activation of several caspases. Inhibition of caspases activity by pan‐caspase inhibitor z‐vad‐fmk completely abrogated the ESOM‐induced cell death. ESOM administration (2.5 mg kg−1) to SCID mice engrafted with human melanoma reduced tumor growth, consistent with decrease of proliferating cells and clear reduction of pH gradients in tumor tissue. Moreover, systemic ESOM administration dramatically increased survival of human melanoma‐bearing animals, in absence of any relevant toxicity. These data show preclinical evidence supporting the use of PPI as novel therapeutic strategy for melanoma, providing the proof of concept that PPI target human melanoma modifying tumor pH gradients.

Proton pump inhibition induces autophagy as a survival mechanism following oxidative stress in human melanoma cells

Proton pump inhibitors (PPI) target tumour acidic pH and have an antineoplastic effect in melanoma. The PPI esomeprazole (ESOM) kills melanoma cells through a caspase-dependent pathway involving cytosolic acidification and alkalinization of tumour pH. In this paper, we further investigated the mechanisms of ESOM-induced cell death in melanoma. ESOM rapidly induced accumulation of reactive oxygen species (ROS) through mitochondrial dysfunctions and involvement of NADPH oxidase. The ROS scavenger N-acetyl-L-cysteine (NAC) and inhibition of NADPH oxidase significantly reduced ESOM-induced cell death, consistent with inhibition of cytosolic acidification. Autophagy, a cellular catabolic pathway leading to lysosomal degradation and recycling of proteins and organelles, represents a defence mechanism in cancer cells under metabolic stress. ESOM induced the early accumulation of autophagosomes, at the same time reducing the autophagic flux, as observed by WB analysis of LC3-II accumulation and by fluorescence microscopy. Moreover, ESOM treatment decreased mammalian target of rapamycin signalling, as reduced phosphorylation of p70-S6K and 4-EBP1 was observed. Inhibition of autophagy by knockdown of Atg5 and Beclin-1 expression significantly increased ESOM cytotoxicity, suggesting a protective role for autophagy in ESOM-treated cells. The data presented suggest that autophagy represents an adaptive survival mechanism to overcome drug-induced cellular stress and cytotoxicity, including alteration of pH homeostasis mediated by proton pump inhibition.

Effect Of Human Natural Killer and γδ T Cells on the Growth of Human Autologous Melanoma Xenografts in SCID Mice

Natural killer (NK) cells were first identified for their ability to kill tumor cells of different origin in vitro. Similarly, γδ T lymphocytes display strong cytotoxic activity against various tumor cell lines. However, the ability of both the NK and γδ cells to mediate natural immune response against human malignant tumors in vivo is still poorly defined. Severe combined immunodeficient (SCID) mice have been successfully engrafted with human tumors. In this study, the antitumor effect of local as well as of systemic treatments based on NK cells or Vδ1 or Vδ2 γ/δ T lymphocytes against autologous melanoma cells was investigated in vivo. The results show that all three of the populations were effective in preventing growth of autologous human melanomas when both tumor and lymphoid cells were s.c. inoculated at the same site. However, when lymphoid cells were infused i.v., only NK cells and Vδ1 γ/δ T lymphocytes could either prevent or inhibit the s.c. growth of autologous melanoma. Accordingly, both NK cells and Vδ1 γδ T lymphocytes could be detected at the s.c. tumor site. In contrast, Vδ2 γδ T lymphocytes were only detectable in the spleen of the SCID mice. Moreover, NK cells maintained their inhibitory effect on tumor growth even after discontinuation of the treatment. Indeed they were present at the tumor site for a longer period. These data support the possibility to exploit NK cells and Vδ1 γδ T lymphocytes in tumor immunotherapy. Moreover, our study emphasizes the usefulness of human tumor/SCID mouse models for preclinical evaluation of immunotherapy protocols against human tumors.

Escape strategies and reasons for failure in the interaction between tumour cells and the immune system: how can we tilt the balance towards immune-mediated cancer control?

The last decade has witnessed an exponential increase in the attempts to demonstrate that adaptive immunity can effectively detect cancer cells and impair their growth invivo in cancer patients. However, clinical trials of immunotherapy with a broad array of immunisation strategies have depicted a rather disappointing scenario, suggesting that successful control of tumour growth by immunotherapeutic treatments may not be an easy task to achieve. The attention of tumour immunologists has thus been switched to the potential reasons of failure, and extensive efforts are being made in defining the cellular and molecular pathways interfering with the capacity of the immune system to develop powerful immunological reactions against tumour cells. Although many of these pathways have been well characterised in murine models, little and controversial information about their role in determining neoplastic progression in cancer patients is available. This d-iscrepancy at the moment represents one of the major limitations in understanding the obstacles to the invivo development of protective Tcell-m-ediated immune responses against tumours, and how pharmacological or biological interventions aimed at bypassing tumour escape mechanisms would indeed result in a clinical benefit. The study of the reasons for the failure of the immune system to control tumour growth, which have to be ascribed to highly interconnected phenomena occurring at both tumour and immune levels, could in the near future provide adequate tools to fight c-ancer by finely tuning the host environment through biological therapies.

Potent phagocytic activity discriminates metastatic and primary human malignant melanomas: a key role of ezrin.

Features of phagocytosis have been observed in human tumors, but the phagocytic apparatus of tumor cells and the mechanism(s) underlying this phenomenon have yet to be defined. To address the phenomenon of phagocytosis, its underlying mechanism(s), and its possible role in tumor biology, we used human melanoma cells as a prototypic model. Our results showed that a process of phagocytosis of apoptotic cells occurs in vivo in human melanoma. This finding was consistent with evidence that human melanoma cells in vitro express all of the known lysosomal and phagocytic markers on their cytoplasmic vesicles and that a process of phagocytosis occurs in these vesicles. However, exclusively human melanoma cells deriving from metastatic lesions possess an efficient phagocytic machinery responsible for a macrophage-like activity against latex beads, yeast, and apoptotic cells of different origins, which was comparable to that of human primary macrophages. Moreover, the actin-binding protein ezrin was expressed on phagocytic vacuoles of melanoma cells and of cells deriving from a human adenocarcinoma; both treatment with cytochalasin B and specific inhibition of ezrin synthesis strongly affected the phagocytic activity of melanoma cells. This suggests that the association with the actin cytoskeleton is a crucial requirement for the development of this phenomenon. Hence our data provide evidence for a potent phagocytic activity exerted by metastatic melanoma cells possibly involved in determining the level of aggressiveness of human melanoma. This suggests that the assessment of phagocytic activity may be exploited as a new tool to evaluate the malignancy of human melanoma. Moreover, our data suggest that gene therapy or drug treatments aimed at inhibiting actin assembly to the phagosomal membranes may be proposed as a new strategy for the control of tumor aggressiveness.

Small interfering RNA targeting the subunit ATP6L of proton pump V-ATPase overcomes chemoresistance of breast cancer cells

One of the mechanisms of multiple drug resistance (MDR) is inappropriate sequestration of basic chemotherapeutic agents in acidic endo-lysosomes of cells. The protonation, sequestration, and secretion (PSS) model indicates that drug distribution can be affected by intracellular pH such as lysosomal pH. The vacuolar-H(+)-ATPase (V-ATPase) plays an important role in regulation of intracellular pH by pumping protons into acidic endosomes via an ATP-driven process. In this study, ATP6L, the 16kDa subunit of V-ATPase, was knocked-down by anti-ATP6L small interfering RNA (siRNA) to study the effect on chemosensitivity in the human drug-resistant breast cancer cells MCF-7/ADR. Introduction of anti-ATP6L small interfering RNA duplex into drug-resistant cancer cells significantly inhibited the expression of ATP6L mRNA and protein, as detected by qRT-PCR and Western blot. Inhibition of ATP6L expression by siRNA in MCF-7/ADR sensitized the cells to the cytotoxicity of basic chemotherapeutic agents like doxorobicin, 5-fluorourocil and vincristine. This effect was mediated by a significant increase in lysosomal pH and retention of anticancer drugs into nuclei of cells. These results support the role of tumor acidity in resistance to chemotherapy and provide a rationale for the use of tumor pH modifier agents as coadjuvants in novel anticancer therapies.

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.