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Activating BRAF or NRAS mutations have been found in 80% of human sporadic melanomas, but only one of these genetic alterations could be detected in each tumour. This suggests that BRAF and NRAS ‘double mutants’ may not provide advantage for tumour growth, or may even be selected against during tumorigenesis. However, by applying mutant-allele-specific-amplification-PCR method to short-term melanoma lines, one out of 14 tumours was found to harbour both BRAFV600E and the activating NRASQ61R mutations. On the other hand, analysis of 21 melanoma clones isolated by growth in soft agar from this tumour indicated that 16/21 clones harboured a BRAFV600E, but were wild-type for NRAS, whereas the remaining had the opposite genotype (NRASQ61R/wild-type BRAF). When compared to BRAFV600E clones, NRASQ61R clones displayed reduced growth in soft agar, but higher proliferative ability in vitro in liquid medium and even in vivo after grafting into SCID/SCID mice. These data suggest that NRAS and BRAF activating mutations can coexist in the same melanoma, but are mutually exclusive at the single-cell level. Moreover, the presence of NRASQ61R or BRAFV600E is associated with distinct in vitro and in vivo growth properties of neoplastic cells.Oncogene (2006) 25, 3357–3364. doi:10.1038/sj.onc.1209379; published online 6 February 2006 [ABSTRACT FROM AUTHOR]

CD95(APO-1/Fas)-mediated apoptosis of bystander uninfected T cells exerts a major role in the HIV-1-mediated CD4+ T-cell depletion. HIV-1 gp120 has a key role in the induction of sensitivity of human lymphocytes to CD95-mediated apoptosis through its interaction with the CD4 receptor. Recently, we have shown the importance of CD95/ezrin/actin association in CD95-mediated apoptosis. In this study, we explored the hypothesis that the gp120-mediated CD4 engagement could be involved in the induction of susceptibility of primary human T lymphocytes to CD95-mediated apoptosis through ezrin phosphorylation and ezrin-to-CD95 association. Here, we show that gp120/IL-2 combined stimuli, as well as the direct CD4 triggering, on human primary CD4+T lymphocytes induced an early and stable ezrin activation through phosphorylation, consistent with the induction of ezrin/CD95 association and susceptibility to CD95-mediated apoptosis. Our results provide a new mechanism through which HIV-1-gp120 may predispose resting CD4+T cell to bystander CD95-mediated apoptosis and support the key role of ezrin/CD95 linkage in regulating susceptibility to CD95-mediated apoptosis.Cell Death and Differentiation (2004) 11, 574-582. doi:10.1038/sj.cdd.4401374 Published online 23 January 2004 [ABSTRACT FROM AUTHOR]

Background: Cancer patients are among the main consumers of traditional, complementary, integrative, and alternative medicine (TCIM) such as natural products (herbals, integrators, etc.) and mind and body practices (yoga, acupuncture, etc.)., Methods: A questionnaire on TCIM was submitted to 415 Italian cancer patients. The questionnaire consisted of three sections: (i) biographical and clinical information; (ii) use of natural substances; and (iii) use of mind-body practices., Results: 406 patients completed the questionnaire. The prevalence of TCIM use was 72.3%. Of them, 75.6% started to use TCIM after a tumor diagnosis. The main reasons for using TCIM were to mitigate side effects (65.0%), to regain physical and mental balance (35.9%), to relieve pain (18.3%), and to improve the efficacy of cancer therapy (16.0%). 44.7% of patients taking natural products used them during conventional therapies (chemotherapy, radiotherapy, etc.), and in 67.5% of cases without consulting a doctor. As a consequence, only about 50% of patients taking natural substances used these compounds appropriately, and the most common errors were related with the purpose of reducing the side effects of the therapy (52.3%) and for boosting immune system (32.1%)., Conclusions: There is an impelling need to provide patients with scientifically validated information to raise awareness about the benefits and risks of using TCIM., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2022 Massimo Bonucci et al.)

Background: The identification of novel biomarkers for the early detection and monitoring of gastric (GC) and colorectal cancer (CRC) is of paramount importance. TM9SF4 is a newly described V-ATPase interacting protein involved in the malignant progression of cancer cells. While TM9SF4 expression pattern and cellular localization have been described in in vitro in tumor cell lines of different histotypes, its expression in gastrointestinal tumor tissues has never been investigated., Methods: In this study, we detected by immunohistochemistry (IHC) in tumor and surrounding healthy tissues TM9SF4, in comparison with clinically adopted biomarkers CEA and CA 19-9 to evaluate TM9SF4 potential as a novel tissue marker for early detection and monitoring of GC and CRC cancers., Results: The expression of TM9SF4, CEA and CA 19-9 was evaluated in samples from 108 cancer patients (68 with GC and 40 CRC) and in healthy tissues from 20 non-cancer patients. Our results clearly suggest that TM9SF4 expression was significantly increased in GC and CRC samples and significantly correlated to disease stage in both cancer types., Conclusions: We propose TM9SF4 as highly specific cancer biomarker, exploitable for disease detection and staging of gastrointestinal cancers patients, with tumor tissue levels of expression outperforming those of clinically adopted markers such as CEA and CA 19-9., Competing Interests: Conflicts of interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/tcr-20-516). Dr. Guazzi reports he is an employee of HansaBioMed OU, a company that sells TM9SF4 antibodies. Dr. Chiesi and Dr. Zarovni report that they are inventors of the patent “Monoclonal antibodies, hybridomas nd methods for use”. Pubbl n. 20120122118 licensed to Exosomics spa and Dr Antonio Chiesi is an employee of HansaBioMed OU (HBM) a company that sells TM9SF4 antibodies. Dr. Lozupone reports he is one inventor of the patent “Monoclonal antibodies, hybridomas nd methods for use”. Pubbl n. 20120122118 licensed to Exosomics Spa. The other authors have nothing to disclose., (2020 Translational Cancer Research. All rights reserved.)

Colorectal cancer (CRC) is the second leading cause of cancer mortality in both men and women worldwide. Survival of patients is significantly associated with disease stage at diagnosis. Recent studies highlighted a role of exosomes in CRC development and progression, thus raising the interest on these nanosized vesicular structures as possible biomarkers. Exosomes contain a large variety of molecules, including proteins, lipids and nucleic acids, that are exchanged between cells either within tumor microenvironment or at distant sites from the primary tumor, where they prepare a suitable soil for tumor metastases. The present review summarizes the principal effects of exosomes on CRC development, progression, and provides an update of the most recent findings on the use of exosomal molecules as diagnostic, prognostic and predictive biomarkers in CRC., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Insulin-like-factor-binding-protein 3 (IGFBP-3) is known to modulate the activity of insulin-like growth factors (IGFs) besides having a number of IGF-independent effects on cell growth and survival. IGFBP-3 has been reported to decrease significantly in the blood serum of patients affected by certain cancers. In the present work, we have evaluated the levels of IGFBP-3 in the blood serum and tissues of patients affected by cutaneous melanoma, showing that loss of IGFBP-3 from both is strongly correlated with disease progression and reduced survival. In vitro treatment with IGFBP-3 of human and murine metastatic melanoma cell lines specifically inhibited the cells' migratory and invasive behaviour, inducing up-regulation of melanocytic differentiation markers such as tyrosinase activity and melanin content. A molecular analysis of the cellular pathways transducing the effect of IGFBP-3 implicated the Akt-GSK3β axis. Moreover, administration of IGFBP-3 in vivo to SCID mice inoculated with human metastatic melanoma cells strongly reduced or completely inhibited tumor growth. In summary, IGFBP-3 appears to exert a specific inhibitory effect on melanoma growth and dissemination, suggesting that it may qualify as a useful therapeutic agent in melanomas and perhaps other cancers, at the least as a valid adjuvant therapy during treatment with conventional anti-tumoral drugs.

Dormancy is defined as a stable but reversible nonreplicating state of Mycobacterium tuberculosis. It is currently thought that dormant M. tuberculosis (D-Mtb) is responsible for latent tuberculosis (TB) infection. Recently, D-Mtb was also shown in sputa of patients with active TB, but the capacity of D-Mtb to stimulate specific immune responses was not investigated. We observed that purified protein derivative-specific human CD4(+) T lymphocytes recognize mycobacterial Ags more efficiently when macrophages are infected with D-Mtb instead of replicating M. tuberculosis (R-Mtb). The different Ag recognition occurs even when the two forms of mycobacteria equally infect and stimulate macrophages, which secrete the same cytokine pattern and express MHC class I and II molecules at the same levels. However, D-Mtb but not R-Mtb colocalizes with mature phagolysosome marker LAMP-1 and with vacuolar proton ATPase in macrophages. D-Mtb, unlike R-Mtb, is unable to interfere with phagosome pH and does not inhibit the proteolytic efficiency of macrophages. We show that D-Mtb downmodulates the gene Rv3875 encoding for ESAT-6, which is required by R-Mtb to block phagosome maturation together with Rv3310 gene product SapM, previously shown to be downregulated in D-Mtb. Thus, our results indicate that D-Mtb cannot escape MHC class II Ag-processing pathway because it lacks the expression of genes required to block the phagosome maturation. Data suggest that switching to dormancy not only represents a mechanism of survival in latent TB infection, but also a M. tuberculosis strategy to modulate the immune response in different stages of TB.

Overexpression of the mdr1 gene encoding P-glycoprotein (Pgp) exerts a major role in reducing the effectiveness of cytotoxic therapy in osteosarcoma. The interaction between actin and Pgp has been shown to be instrumental in the establishment of multidrug resistance (MDR) in human tumor cells. The cytoskeleton linker ezrin exerts a pivotal role in maintaining the functional connection between actin and Pgp. We investigated the role of ezrin in a human multidrug-resistant osteosarcoma cell line overexpressing Pgp and compared it to its counterpart that overexpresses an ezrin deletion mutant. The results showed that Pgp binds at amino acid residues 149-242 of the N-terminal domain of ezrin. The interaction between ezrin and Pgp occurs in the plasma membrane of MDR cells, where they also co-localize with the ganglioside G(M1) located in lipid rafts. The overexpression of the ezrin deletion mutant entirely restored drug susceptibility of osteosarcoma cells, consistent with Pgp dislocation to cytoplasmic compartments and abrogation of G(M1) /Pgp co-localization at the plasma membrane. Our study provides evidence that ezrin exerts a key role in MDR of human osteosarcoma cells through a Pgp-ezrin-actin connection that is instrumental for the permanence of Pgp into plasma membrane lipid rafts. We also show for the first time that Pgp-binding site is localized to amino acid residues 149-242 of the ezrin Band 4.1, Ezrin/Radixin/Moesin (FERM) domain, thus proposing a specific target for future molecular therapy aimed at counteracting MDR in osteosarcoma patients., (Copyright © 2011 UICC.)

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.

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.

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.

The membrane cytoskeleton cross-linker, ezrin, has recently been depicted as a key regulator in the progression and metastasis of several pediatric tumors. Less defined appears the role of ezrin in human adult tumors, especially melanoma. We therefore addressed ezrin involvement in the metastatic phenotype of human adult metastatic melanoma cells. Our results show that cells resected from melanoma metastatic lesions of patients, display marked metastatic spreading capacity in SCID mice organs. Stable transfection of human melanoma cells with an ezrin deletion mutant comprising only 146 N-terminal aminoacids led to the abolishment of metastatic dissemination. In vitro experiments revealed ezrin direct molecular interactions with molecules related to metastatic functions such as CD44, merlin and Lamp-1, consistent with its participation to the formation of phagocitic vacuoles, vesicular sorting and migration capacities of melanoma cells. Moreover, the ezrin fragment capable of binding to CD44 was shorter than that previously reported, and transfection with the ezrin deletion mutant abrogated plasma membrane Lamp-1 recruitment. This study highlights key involvement of ezrin in a complex machinery, which allows metastatic cancer cells to migrate, invade and survive in very unfavorable conditions. Our in vivo and in vitro data reveal that ezrin is the hub of the metastatic behavior also in human adult tumors., (Copyright 2008 UICC.)

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.

Proton pumps like the vacuolar-type H+ ATPase (V-ATPase) are involved in the control of cellular pH in normal and tumor cells. Treatment with proton pump inhibitors (PPI) induces sensitization of cancer cells to chemotherapeutics via modifications of cellular pH gradients. It is also known that low pH is the most suitable condition for a full PPI activation. Here, we tested whether PPI treatment in unbuffered culture conditions could affect survival and proliferation of human B-cell tumors. First, we showed that PPI treatment increased the sensitivity to vinblastine of a pre-B acute lymphoblastic leukemia (ALL) cell line. PPI, per se, induced a dose-dependent inhibition of proliferation of tumor B cells, which was associated with a dose- and time-dependent apoptotic-like cytotoxicity in B-cell lines and leukemic cells from patients with pre-B ALL. The effect of PPI was mediated by a very early production of reactive oxygen species (ROS), that preceded alkalinization of lysosomal pH, lysosomal membrane permeabilization, and cytosol acidification, suggesting an early destabilization of the acidic vesicular compartment. Lysosomal alterations were followed by mitochondrial membrane depolarization, release of cytochrome c, chromatin condensation, and caspase activation. However, inhibition of caspase activity did not affect PPI-induced cell death, whereas specific inhibition of ROS by an antioxidant (N-acetylcysteine) significantly delayed cell death and protected both lysosomal and mitochondrial membranes. The proapoptotic activity of PPI was consistent with a clear inhibition of tumor growth following PPI treatment of B-cell lymphoma in severe combined immunodeficient mice. This study further supports the importance of acidity and pH gradients in tumor cell homeostasis and suggests new therapeutic approaches for human B-cell tumors based on PPI.

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.

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 in vivo 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 discrepancy at the moment represents one of the major limitations in understanding the obstacles to the in vivo development of protective T cell-mediated 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 cancer by finely tuning the host environment through biological therapies.

Background: Resistance to antitumor agents is a major cause of treatment failure in patients with cancer. Some mechanisms of tumor resistance to cytotoxic drugs may involve increased acidification of extracellular compartments. We investigated whether proton pump inhibitors (PPIs), currently used in the anti-acid treatment of peptic disease, could inhibit the acidification of the tumor microenvironment and increase the sensitivity of tumor cells to cytotoxic agents., Methods: We pretreated cell lines derived from human melanomas, adenocarcinomas, and lymphomas with the PPIs omeprazole, esomeprazole, or pantoprazole and tested their response to cytotoxic drugs in cell death assays. We also evaluated extracellular and intracellular pH and vacuolar-H+-ATPase (V-H+-ATPase) expression, distribution, and activity in PPI-pretreated cells by using western blot analyses, immunocytochemistry, laser scanning confocal analysis, and bioluminescence assays. Finally, we evaluated human melanoma growth and cisplatin sensitivity with or without omeprazole pretreatment in xenografted SCID/SCID mice., Results: PPI pretreatment sensitized tumor cell lines to the effects of cisplatin, 5-fluorouracil, and vinblastine, with an IC50 value reduction up to 2 logs. PPI pretreatment was associated with the inhibition of V-H+-ATPase activity and increases in both extracellular pH and the pH of lysosomal organelles. PPI pretreatment induced a marked increase in the cytoplasmic retention of the cytotoxic drugs, with clear targeting to the nucleus in the case of doxorubicin. In in vivo experiments, oral pretreatment with omeprazole was able to induce sensitivity of human solid tumors to cisplatin., Conclusion: Our results open new possibilities for the treatment of drug-resistant tumors through combination strategies based on the use of well-tolerated pH modulators such as PPIs.

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.

Natural killer (NK) cells were first identified for their ability to kill tumor cells of different origin in vitro. Similarly, gammadelta T lymphocytes display strong cytotoxic activity against various tumor cell lines. However, the ability of both the NK and gammadelta 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 Vdelta1 or Vdelta2 gamma/delta 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 Vdelta1 gamma/delta T lymphocytes could either prevent or inhibit the s.c. growth of autologous melanoma. Accordingly, both NK cells and Vdelta1 gammadelta T lymphocytes could be detected at the s.c. tumor site. In contrast, Vdelta2 gammadelta 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 Vdelta1 gammadelta 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.

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.

The identification of appropriate mouse models could be useful in carefully evaluating the actual role of the in vivo development of antigen-loss variants during antigen-specific vaccine therapy of human tumors. In this study we investigated the level of efficacy of a MART-1/Melan-A-specific CD8+ T cell clone against its autologous melanoma in a severe combined immunodeficiency (SCID) mouse model, in which the tumor cells expressed in vivo heterogeneous and suboptimal levels of MART-1. The subcutaneous co-injection of the MART-1/Melan-A-reactive T cell clone A42 with MART-1/Melan-A+ autologous human melanoma cells into SCID mice caused a total inhibition of tumor growth. However, the systemic treatment with A42 clone lymphocytes resulted in only 50-60% inhibition of tumor growth, although the T cell clone targeted the tumors and the MART-1+ cells virtually disappeared from the tumors. This study suggests that an immunotherapy based on the expansion of an antigen-specific T cell clone generated in vitro is highly efficient in abolishing tumor growth when the target antigen is fully expressed, but leads to in vivo immunoselection of antigen-loss variants in the presence of suboptimal levels of antigen expression. Furthermore, this work shows that human tumors/SCID mouse models may be useful in evaluating the in vivo efficacy of adoptive immunotherapies.

Cytoskeleton plays a crucial role in natural killer cell function. In this study the expression and subcellular distribution of ezrin, radixin and moesin, a family of proteins that connect actin filaments to many membrane structures, were evaluated in human NK cells. The results showed that NK cells expressed all these proteins, while NK cell-deprived peripheral blood leukocytes and purified T lymphocytes did not express radixin. Only ezrin changed its distribution following IL-2 activation and all three ezrin, moesin and radixin were polarized on uropods of adherent natural killer cells. Ezrin and radixin co-localized with the perforin granules at the intimate sites of contact between NK and the target cells, while moesin remains uniformly distributed on the membrane of NK cells. Ezrin, radixin and perforin co-localization was undetected in non-lytic conjugates and inhibited by treatment with actin depolymerizing agents. These results suggest that ezrin and radixin may exert a role in NK activity, particularly in the trafficking of perforin granules to the NK/target cells contact site. Moreover, our data suggest that radixin may represent an additional biological marker of human NK cells and that this protein may hold a specific role in NK cell function.

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.

The c-Met tyrosine kinase receptor and its ligand, Hepatocyte Growth Factor/ Scatter Factor, have been implicated in human cancer. We have previously described that the transgenic expression of a truncated form of human c-Met (cyto-Met) in the liver confers resistance to several apoptotic stimuli. Here we show the impact of cyto-Met expression on liver proliferation and transformation. Despite a sixfold increase of hepatocyte proliferation, adult transgenic livers displayed normal size and architecture. We present evidence showing that activation of TGF-beta1 signalling controls the liver mass in cyto-Met mice. The oncogenic potential of cyto-Met was further assessed in the context of c-Myc-induced hepatocarcinogenesis, using WHV/c-Myc transgenic mice. Co-expression of cyto-Met and c-Myc further enhanced hepatocyte proliferation and caused a dramatic acceleration of the Myc-induced tumorigenesis, leading to the emergence of hepatocarcinomas in 3-4-month-old animals. Importantly, the TGF-beta receptor type II expression was strongly downregulated in most tumours, indicating that impairment of TGF-beta1-mediated growth inhibition plays a major role in accelerated neoplastic development. The strong potential of cyto-Met for oncogenic cooperation without direct transforming activity designates cyto-Met mice as an ideal tool for studying the early steps of multistage hepatocarcinogenesis and for identification of prognostic markers of transformation.

P-glycoprotein is a 170-kd glycosylated transmembrane protein, expressed in a variety of human cells and belonging to the adenosine triphosphate-binding cassette transporter family, whose membrane expression is functionally associated with the multidrug resistance phenotype. However, the mechanisms underlying the regulation of P-glycoprotein functions remain unclear. On the basis of some evidence suggesting P-glycoprotein-actin cytoskeleton interaction, this study investigated the association of P-glycoprotein with ezrin, radixin, and moesin, a class of proteins that cross-link actin filaments with plasma membrane in a human cell line of lymphoid origin and that have been shown to link other ion-pump-related proteins. To this purpose, a multidrug-resistant variant of CCRF-CEM cells (CEM-VBL100) was used as a model to investigate the following: (1) the cellular localizations of P-glycoprotein and ezrin, radixin, and moesin and their molecular associations; and (2) the effects of ezrin, radixin, and moesin antisense oligonucleotides on multidrug resistance and P-glycoprotein function. The results showed that: (1) P-glycoprotein colocalized and coimmunoprecipitated with ezrin, radixin, and moesin; and (2) treatment with antisense oligonucleotides for ezrin, radixin, and moesin restored drug susceptibility consistently with inhibition of both drug efflux and actin-P-glycoprotein association and induction of cellular redistribution of P-glycoprotein. These data suggest that P-glycoprotein association with the actin cytoskeleton through ezrin, radixin, and moesin is key in conferring to human lymphoid cells a multidrug resistance phenotype. Strategies aimed at inhibiting P-glycoprotein-actin association may be helpful in increasing the efficiency of both antitumor and antiviral therapies.

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.

Severe combined immunodeficient (SCID) mice generally do not reject allogeneic or xenogeneic organ grafts and represent a unique model for investigating in vivo the behaviour of both normal and neoplastic human cells. However, cells from human primary tumors often do not grow in SCID mice. We have previously shown that the major reaction of SCID mice to the engraftment of human peripheral blood leukocytes is a massive granulocyte recruitment into the site of transplantation. In this study, we have investigated the role of murine granulocytes in the control of human tumor cell growth in SCID mice. We report here that murine granulocytes infiltrate and delimit the human tumor mass and that treatment of SCID mice with anti-murine granulocyte antibody markedly improves the growth of human tumor cell lines of different origin through suppression of the host granulocyte reaction. This finding provides a new tool for improving the human tumor take in SCID mice, thus opening new perspectives for a practical in vivo preclinical test of anti-tumor strategies. Moreover, this study, even with the limits of the known natural reaction against xenotransplants, further supports the importance of granulocytes in the control of tumor take and growth., (Copyright 2000 Wiley-Liss, Inc.)