Hannah Rabinowich

Lymphocyte apoptosis induced by Fas ligand- expressing ovarian carcinoma cells. Implications for altered expression of T cell receptor in tumor-associated lymphocytes.

We have recently reported that tumor-associated lymphocytes obtained from ascitic fluids of women with ovarian carcinoma (OvCA) demonstrate a marked decrease in expression of cytoplasmic CD3-zeta and surface CD3-epsilon chains, which is associated with altered function of T cell receptor (TcR). We now demonstrate that OvCAs in situ and in culture express functional Fas ligand (FasL), capable of triggering an intrinsic cell death program in Fas-expressing T cells. The possibility of a relationship between cell death and altered expression of TcR was examined. The data indicate that alterations in expression of CD3-zeta and CD3-epsilon chains in T cells coincubated with OvCA are related to tumor-induced apoptosis, as the addition of pan-caspase inhibitors, DEVD-cho or YVAD-cho, prevents both the in vitro induction of T cell death by OvCA cells and the changes in the level of expression of CD3-zeta and CD3-epsilon chains. In the presence of Fas-Fc fusion protein, but not Fc-control protein, the loss in expression of CD3-zeta and CD3-epsilon chains induced in T cells by FasL+ OvCA cells was prevented. These results suggest that the loss in expression of CD3-zeta and CD3-epsilon chains in T lymphocytes interacting with OvCA cells is associated with apoptosis mediated by FasL-expressing tumor cells.

Autophagic degradation of active caspase-8: A crosstalk mechanism between autophagy and apoptosis

Apoptotic defects endow tumor cells with survival advantages. Such defects allow the cellular stress response to take the path of cytoprotective autophagy, which either precedes or effectively blocks an apoptotic cascade. Inhibition of the cytoprotective autophagic response shifts the cells toward apoptosis, by interfering with an underlying molecular mechanism of cytoprotection. The current study has identified such a mechanism that is centered on the regulation of caspase-8 activity. The study took advantage of Bax-/- Hct116 cells that are TRAIL-resistant despite significant DISC processing of caspase-8, and of the availability of a caspase-8-specific antibody that exclusively detects the caspase-8 large subunit or its processed precursor. Utilizing these biological tools, we investigated the expression pattern and subcellular localization of active caspase-8 in TRAIL-mediated autophagy and in the autophagy-to-apoptosis shift upon autophagy inhibition. Our results suggest that the TRAIL-mediated autophagic response counter-balances the TRAIL-mediated apoptotic response by the continuous sequestration of the large caspase-8 subunit in autophagosomes and its subsequent elimination in lysosomes. The current findings are the first to provide evidence for regulation of caspase activity by autophagy and thus broaden the molecular basis for the observed polarization between autophagy and apoptosis.

Interrelated roles for Mcl-1 and BIM in regulation of TRAIL-mediated mitochondrial apoptosis.

The current study demonstrates a novel cross-talk mechanism between the TRAIL receptor death signaling pathway and the mitochondria. This newly identified pathway is regulated at the mitochondrial outer membrane by a complex between the prosurvival Bcl-2 member, Mcl-1 and the BH3-only protein, Bim. Under non-apoptotic conditions, Bim is sequestered by Mcl-1. Direct degradation of Mcl-1 by TRAIL-activated caspase-8 or caspase-3 produces Mcl-1-free Bim that mediates a Bax-dependent apoptotic cascade. Using Mcl-1 or Bim RNAi, we demonstrate that a loss in Mcl-1 expression significantly enhances the mitochondrial apoptotic response to TRAIL that is now mediated by freed Bim. Whereas overexpression of Mcl-1 contributes to the preservation of the mitochondrial membrane potential, Mcl-1 knockdown facilitates the Bim-mediated dissipation of this potential. Loss of Mcl-1 contributes to an increased level of caspase activity downstream of the mitochondrial response to TRAIL. Furthermore, the Mcl-1 expression level at the mitochondrial outer membrane determines the release efficiency for the apoptogenic proteins cytochrome c, Smac, and HtrA2 in response to Bim. These are the first findings to demonstrate the involvement of Bim in the TRAIL-mediated mitochondrial cascade. They also suggest that Mcl-1 may serve as a direct substrate for TRAIL-activated caspases implying the existence of a novel TRAIL/caspase-8/Mcl-1/Bim communication mechanism between the extrinsic and the intrinsic apoptotic pathways.

Involvement of Protective Autophagy in TRAIL Resistance of Apoptosis-defective Tumor Cells

Targeting TRAIL receptors with either recombinant TRAIL or agonistic DR4- or DR5-specific antibodies has been considered a promising treatment for cancer, particularly due to the preferential apoptotic susceptibility of tumor cells over normal cells to TRAIL. However, the realization that many tumors are unresponsive to TRAIL treatment has stimulated interest in identifying apoptotic agents that when used in combination with TRAIL can sensitize tumor cells to TRAIL-mediated apoptosis. Our studies suggest that various apoptosis defects that block TRAIL-mediated cell death at different points along the apoptotic signaling pathway shift the signaling cascade from default apoptosis toward cytoprotective autophagy. We also obtained evidence that inhibition of such a TRAIL-mediated autophagic response by specific knockdown of autophagic genes initiates an effective mitochondrial apoptotic response that is caspase-8-dependent. Currently, the molecular mechanisms linking disabled autophagy to mitochondrial apoptosis are not known. Our analysis of the molecular mechanisms involved in the shift from protective autophagy to apoptosis in response to TRAIL sheds new light on the negative regulation of apoptosis by the autophagic process and by some of its individual components.

The role of Fas/FasL in immunosuppression induced by human tumors

The issue of tumor-induced immunosuppression has been debated for many years [25, 25, 56, 72]. The mechanisms that might be responsible for this type of immune dysfunction in tumor-bearing individuals have not been clearly defined, however, and the variable levels of systemic immunosuppression (from none to a complete anergy) observed in dozens of studies performed in cancer patients have been difficult to explain in a rational way. Nevertheless, the presence of local, and often systemic, defects in lymphocyte functions of patients with advanced malignancies has been a recurrent theme in cancer immunology. Tumor-induced immunosuppression has been used as an explanation for the lack of responses to cancer therapies aimed at the up-regulation of the immune system in these patients [25, 25, 56, 72]. The lack of correlations between tumor regression and antitumor T cell (cytotoxic T lymphocyte CTL) responses encountered in recent vaccine trials suggests that the adverse effects of tumors on the host may be difficult to overcome [8, 29]. Similarly, the absence or low level of CTL seen in cancer patients immunized with tumor vaccines suggests that either immune effector cells are not generated or that they are generated but do not survive in vivo. In either scenario, the result is an inadequate antitumor response, and it is reasonable to speculate that the patients with particularly immunosuppressive tumors have a worse prognosis compared to the patients whose immune system is relatively intact. This then suggests that mutual interactions between the host immune system and the tumor are important for the disease outcome, and that it might be possible to shift the balance of these interactions in favor of the host by therapeutic interventions. Before this can be accomplished, however, it is first necessary to recognize the problem, that is to admit that human tumors indeed can and do induce immune cell dysfunction and even death, and second, to investigate the mechanisms responsible for it. In this review, we will focus on one such recently discovered mechanism, namely, the Fas/FasL pathway [50] and propose to illustrate how it could contribute to what has been described as a acounterattacko of tumors against the immune system of the host [53]. Briefly, Fas (CD 95) is a member of the tumor necrosis factor (TNF) family. It is widely expressed on many different cell types. Binding of Fas ligand (FasL) to Fas induces apoptosis in Fas-positive targets [50]. Recently, expression of FasL on the surface as well as in the cytoplasm of human tumor cells has been described [22, 53, 59]. It has also been confirmed that activated lymphocytes that accumulate in human tumors express Fas, and that apoptosis of lymphocytes in situ is a common characteristic of human tumors [59, 60]. These observations led to a hypothesis that FasL+ tumors can induce dysfunction and death of infiltrating lymphocytes, and that tumor-associated immunosuppression results, in part, from death of immune cells in the tumor microenvironment. While this review features the Fas/FasL pathway as a mechanism of tumor-induced apoptosis of immune cells, it is necessary to bear in mind that this may not be the only or even the predominant mechanism operative at the tumor site.

Tumor's other immune targets: dendritic cells.

The induction of apoptosis in T cells is one of several mechanisms by which tumors escape immune recognition. We have investigated whether tumors induce apoptosis in dendritic cells (DC) by co‐culture of murine or human DC with different tumor cell lines for 4–48 h. Analysis of DC morphological features, JAM assay, TUNEL, caspase‐3‐like and transglutaminase activity, Annexin V binding, and DNA fragmentation assays revealed a time‐ and dose‐dependent induction of apoptosis in DC by tumor‐derived factors. This finding is both effector and target specific. The mechanism of tumor‐induced DC apoptosis involved regulation of Bcl‐2 and Bax expression. Double staining of both murine and human tumor tissues confirmed that tumor‐associated DC undergo apoptotic death in vivo. DC isolated from tumor tissue showed significantly higher levels of apoptosis as determined by TUNEL assay when compared with DC isolated from spleen. These findings demonstrate that tumors induce apoptosis in DC and suggest a new mechanism of tumor escape from immune recognition. DC protection from apoptosis will lead to improvement of DC‐based immunotherapies for cancer and other immune diseases. J. Leukoc. Biol. 66: 336–344; 1999.

Expression of cytokine genes or proteins and signaling molecules in lymphocytes associated with human ovarian carcinoma

We have reported that tumor‐associated T or natural killer (NK) lymphocytes purified from ascites of women with ovarian carcinoma show defective expression and function of signaling proteins, including reduced expression of TcR‐ζ chains and p56lck. In this study, the cytokine profiles of both tumor cells and tumor‐associated lymphocytes (TAL) recovered from the tumor milieu were examined. Expression of cytokine genes was studied by semi‐quantitative RT‐PCR and Southern hybridization, and the presence of intracellular cytokine proteins was confirmed by immunostaining. Levels of mRNA encoding the cytokine genes typically transcribed in activated T lymphocytes, including IFN‐γ, IL‐2 and IL‐4, were markedly reduced, as was expression of the corresponding proteins, in TAL‐T or TAL‐NK cells relative to normal PBL‐T or PBL‐NK cells, respectively. Levels of TGF‐β and IL‐6 were unaltered, while those of IL‐10 were up‐regulated. Although both tumor cells and TALs contributed to the enhanced level of IL‐10 expression, a higher proportion of TAL‐T lymphocytes than normal PBL‐T cells expressed IL‐10 protein. The altered profile of cytokine genes and proteins in TALs, TAL‐T or TAL‐NK cells was associated with impaired expression and/or function of signaling molecules, ζ chain and p56lck. Our data suggest that abnormalities in signal transduction commonly seen in lymphocytes obtained from the tumor micro‐environment are related to the concomitantly observed altered patterns of expression of cytokine transcripts and proteins.

Degradation of Mcl-1 by Granzyme B IMPLICATIONS FOR Bim-MEDIATED MITOCHONDRIAL APOPTOTIC EVENTS

Recent studies have suggested that in the absence of Bid, granzyme B (GrB) can utilize an unknown alternative pathway to mediate mitochondrial apoptotic events. The current study has elucidated just such a pathway for GrB-mediated mitochondrial apoptotic alterations. Two Bcl-2 family members have been identified as interactive players in this newly discovered mitochondrial response to GrB: the pro-survival protein Mcl-1L and the pro-apoptotic protein, Bim. Expression of Mcl-1L, which localizes mainly to the outer mitochondrial membrane, decreases significantly in cells subjected to CTL-free cytotoxicity mediated by a combination of GrB and replication-deficient adenovirus. The data suggest that Mcl-1L is a substrate for GrB and for caspase-3, but the two enzymes appear to target different cleavage sites. The cleavage pattern of endogenous Mcl-1L resembles that of in vitro translated Mcl-1L subjected to similar proteolytic activity. Co-immunoprecipitation experiments performed with endogenous as well as with in vitro translated proteins suggest that Mcl-1L is a high affinity binding partner of the three isoforms of Bim (extra-long, long, and short). Bim, a BH3-only protein, is capable of mediating the release of mitochondrial cytochrome c, and this activity is inhibited by the presence of exogenous Mcl-1L. The findings presented herein imply that Mcl-1L degradation by either GrB or caspase-3 interferes with Bim sequestration by Mcl-1L.

Expression of X-linked inhibitor-of-apoptosis protein in hepatocellular carcinoma promotes metastasis and tumor recurrence.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide. Despite significantly improved diagnosis and treatment in recent years, the long‐term therapeutic effect is compromised by the frequent recurrence and metastasis, of which the molecular mechanisms are not fully understood. Our initial studies in established HCC cell lines with different metastatic capabilities indicated a correlation of metastasis with the resistance to apoptosis and therefore the ability to survive in stressed conditions. Subsequent investigation revealed that increased expression of X‐linked inhibitor‐of‐apoptosis protein (XIAP) was correlated with the resistance to apoptosis and enhanced invasiveness in vitro, which could contribute to increased metastatic foci in vivo. Furthermore, we found that nearly 90% of clinical samples from advanced HCC patients expressed high levels of XIAP. Patients with XIAP‐positive tumors had a significantly increased risk of relapse, which resulted from metastasis after total liver resection and orthotopic liver transplantation. Indeed, XIAP expression could be an independent prognostic factor for predicting disease‐free survival rate and overall survival rate of these patients. XIAP expression was also highly correlated with advanced cases that exceeded the Milan criteria and could be a prognostic factor for disease‐free survival in these patients as well. Conclusion: Our studies have shown an important molecule in controlling HCC metastasis, defined a biomarker that can be used to predict HCC recurrence and patient survival after treatment, and suggest that XIAP can be a molecular target subject to intervention to reduce metastasis and recurrence. (HEPATOLOGY 2008;48:497–507.)

Cytolytic cells induce HMGB1 release from melanoma cell lines

High mobility group box 1 (HMGB1) is one of the recently defined damage‐associated molecular pattern molecules, passively released from necrotic cells and secreted by activated macrophage/monocytes. Whether cytolytic cells induce HMGB1 release from tumor cells is not known. We developed a highly sensitive method for detecting intracellular HMGB1 in tumor cells, allowing analysis of the type of cell death and in particular, necrosis. We induced melanoma cell death with cytolytic lymphokine‐activated killing (LAK) cells, tumor‐specific cytolytic T lymphocytes, TRAIL, or granzyme B delivery and assessed intracellular HMGB1 retention or release to investigate the mechanism of HMGB1 release by cytolytic cells. HMGB1 release from melanoma cells (451Lu, WM9) was detected within 4 h and 24 h following incubation with IL‐2‐activated PBMC (LAK activity). HLA‐A2 and MART1 or gp100‐specific cytolytic T lymphocytes induced HMGB1 release from HLA‐A2‐positive and MART1‐positive melanoma cells (FEM X) or T2 cell‐loaded, gp100‐specific peptides. TRAIL treatment, however, induced HMGB1 release, and it is interesting that this extrinsic pathway‐mediated cell death was blocked with the pancaspase inhibitor N‐benzyloxycarbonyl‐Val‐Ala‐Asp‐fluoromethylketone. Conversely, granzyme B delivery did not induce HMGB1 release. HMGB1, along with other intracellular factors released from tumor cells induced by cytolysis, may be important components of the disordered tumor microenvironment. This has important implications for the immunotherapy of patients with cancer. Specifically, HMGB1 may promote healing or immune reactivity, depending on the nature of the local inflammatory response and the presence (or absence) of immune effectors.