Role of Transglutaminase 2 in immunogenic and ER-stress induced cell death in cancer

The objective of an effective therapy against cancer is to destroy every single cancerous cell, including cancer stem cells, in order to allow the survival of the patient. Some anti-cancer therapies tend to “educate” the host’s immune system to recognize the remaining tumour cells, in order to minimize the risk of relapse. Chemotherapy and immunotherapy are often hardly compatible. In fact, most of the chemotherapeutic agents induce DNA damage, in order to trigger apoptosis of the cancer cells. These treatments often result into the induction of massive immune system effector’s depletion. Etoposide and mitomycin C, two widely used drugs, induce non-immunogenic cell death while, other compounds, such as anthracyclines, are able to induce immunogenic cell death. In addition, there are reports suggesting that anthracyclines are able to induce immunogenic apoptosis, have a direct effect on cancer and also they are able to mediate the immune response. It has been demonstrated that those compounds are able to stimulate the exposure of Calreticulin (CRT) on the plasma membrane of tumour cells in a pre-apoptotic step. One of the peculiar biochemical aspects of the immunogenic cell death is this pre-apoptotic translocation of CRT from the Endoplasmic reticulum (endo-CRT) to the cell surface (ecto-CRT). Calreticulin is a Ca2+-binding molecular chaperone expressed in the endoplasmic reticulum, where it takes part in calcium homeostasis. It can be found also in other cellular compartments, such as the nucleus and the plasma membrane, and it is a multi functional protein able to interact with the α-subunit of integrins and with proteins involved in the ER-stress response, such as PDI and ERp57. In addition, it has been shown that CRT expression on the surface of damaged cells might function as an “eat-me” signal, which elicits cellular recognition and removal by macrophages or dendritic cell. Type 2 Transglutaminase (TG2) belongs to a family of Ca2+-dependent enzymes capable of covalently modifying proteins by cross-linking them via the formation of ε(γ-glutamyl)lysine bonds. TG2 is a peculiar member of the family, which could be externalized on the cell surface and thus it mediates the interaction of integrins with fibronectin and cross-links proteins of the extra-cellular matrix. On the basis of its sub-cellular localisation TG2 may also act as a protein disulphide isomerase (PDI) at mitochondrial level or as a G-protein at plasma membrane level. In fact, it has been shown that α-subunit of the etherotrimeric G-proteins is TG2. Interestingly, it has been have shown that the β-subunit of these G-proteins is CRT. CRT interacts with TG2, when TG2 bounds GDP. We hypothesised that TG2-CRT interaction might modulate not only their intra-cellular activities but also their relationships with the plasma-membrane and, possibly, CRT exposition on cell surface. In order to address our hypothesis we assessed the presence of CRT on the plasma membrane of the human neuroblastoma cell line Sk-n-BE(2), which does not express detectable levels of TG2, in respect to the TGA cell line, transfected to achieve high TG2 expression levels. We used different approaches, such as flow cytometry, surface protein purification, to analyse cell surface exposition of CRT in these two cell lines. Our results indicate that, at steady state, the SK-n-BE(2) cell line express about 2 fold more CRT on cell surface, as compared to TGA cells, thus suggesting a role for TG2 in CRT exposure. In addition, we showed that pre-treatment of the cells with cystamnine, a pan inhibitor of TG2 transamidasic activity, lowered the amount of cell surface exposed CRT. On the basis of these results we hypothesised that TG2, when bound to GDP and acting as a G-protein, might bind CRT and prevent its translocation on cell surface. TG2-dependent modulation of CRT translocation is detectable even upon anthracyclines treatment. In fact, when we analysed the presence of CRT on cell surface after doxorubicin treatment, we spotted the same differences detected at steady state. This aspect might be of relevance, as doxorubicin not only induces high level of CRT exposure but also has been show to induce apoptosis and that TG2 might be involved in the modulation of this process (Fesus and Szondy, 2005). It is know that anthracyclines induce ER-stress. On the basis of this evidence we would like to assess whether the various TG2’s biological activities could be involved in cell death regulation cancer. During the last years, many groups demonstrated that TG2 can not only carry out different activities in relation to its sub-cellular localisation (Jones et al., 1997; Malorni et al., 2009; Rodolfo et al., 2004; Siegel et al., 2008) but also that different localisation of the enzyme may result in both pro-survival or pro-death action (Fesus and Szondy, 2005). We previously showed that TG2 may localise on mitochondria, where it’s involved in the maintenance of the organelle’s homeostasis and in the regulation of the mitochondrial pathway of apoptosis. Mitochondria have also a prominent role in the decoying and integration of cellular stress signals, such as DNA damage and ER-stress, and anthracycline, like other chemotherapeutic drugs, are not only DNA damaging agents but also powerful induces of ER-stress. This side of the picture might be very relevant to our project as CRT is an ER resident protein, involved in the activation of the ER-stress response. In addition, during metastasis formation, tumour’s cells are subjected to high levels of stress that involves both ER and mitochondria. We then decided to investigate the possible involvement of TG2 in the overall regulation of ER-stress induced cell death, in our neuroblastoma cell model, by treating cells with two different compounds: tunicamycin and thapsigargin. The choice of these drugs has been dictated by their different modes of action. In fact thapsigargin, is a specific inhibitor of SERCA ATPase (Sarco/endoplasmic reticulum Ca2+ ATPase) and causes the depletion of Ca2+ stores from the endoplasmic reticulum and the increase of the cytosolic Ca2+ concentration. Tunicamycin is an inhibitor of one of major post-translational modifications that take place in the endoplasmic reticulum, the protein’s glycosylation process. We started our investigation by performing time and dose response curve, for the two drugs, and analysing cell death induction by means of flow cytometry. After treatment with 4 and 8 µg/ml of tunicamycin for 24 and 48h, the TGA cell line seems to be less sensitive to cell death induction as compared to the SK-n-BE(2) cell line, thus suggesting TG2 over-expression as protective against tunicamycin induced cell death. Treatments were also carried out with thapsigargin at 2 and 4 µg/ml for the same times. After 24h of treatment there are no differences in cell death levels between the two cell lines for both the doses used while, after 48 h of treatment, there is a clear protection of TGA cells at the highest dose we used. These data suggest TG2 over-expression as protective against thapsigargin induced cell death, and highlighted a dose-dependent effect. The results obtained with thapsigargin were somehow un-expected because thapsigargin causes an increase in the cytosolic calcium concentration, that we hypothesised to be high enough to activate the TG2 transamidasic activity and then to induce apoptosis. We then decided to investigate whether the two cell lines displayed a different response to the ER-stress. To this aim we performed western blot analysis of the expression of ER-stress induced factors, like GRP78 protein, and of cell death markers, like PARP. The results obtained demonstrated that there were no differences between the two cell lines despite the inducer used. As we do not see any difference in the upstream regulation of cellular response to ER-stress, we decided to investigate whether there was any difference in the induction of cell death. It is well known that the ER-stress dependent induction of apoptosis relies on the activation of the pro-apoptotic members of the Bcl-2 family Bax and Bak, and on their action on mitochondrial and ER membranes. In addition, the Ca2+ release induced by Thapsigargin might have an effect on both TG2 activation and mitochondrial release of cytochrome c. In keeping with these considerations, we decided to analyse what happens in the two cell lines after treatment with 4 µg/ml of thapsigargin for 48 hours, by means of an immuno-fluorescence approach. The results obtained showed that cytochrome c release and caspase-3 activation seem to happen at the same extent in the two cell lines. The only difference that we might spot is on the number of fragmented nuclei we observed, again confirming a protection of the TGA cells. Besides these observation it remains still unclear how TG2 over-expression might protect cells from thapsigargin induced cell death. The data obtained by this kind of approach were purely descriptive and not quantitative, so we decided to investigate these events by means of western blot analysis. To this aim we treated the cells in the same way as before and we performed sub-cellular fractionation in order to obtain cytosolic and mitochondrial fraction. The western blot analysis of PARP cleavage, a marker of cell death, revealed a more extensive processing of this protein in the SK-n-BE(2) cell line, in respect to the TGA. This result indicates a more proneness of these cells to cell death induction after thapsigargin treatment, in keeping with what we previously observed by FACS analysis. The analysis of Bax and Bak activation and translocation as well as of the cytochrome c release, does not revealed, in the two cell lines, such striking differences to justify the less sensitivity of the TGA cell line to cell death. On the contrary, the TGA cell lines showed an even marked translocation of Bax and Bak to the mitochondria, suggesting a massive induction of cell death. These results indicates that, upon thapsigargin treatment, the onset of the ER-stress response and the induction of cell death takes places in a similar way in the two cell lines and they does not justify the minor sensitivity displayed by the TGA cell lines. Recently, in vitro experiments have demonstrated that caspase-3 might be a protein substrate for the cross-linking activity of TG2. Following thapsigargin treatment, the increase of the cytosolic Ca2+ concentration activates TG2, which might act on caspase-3 leading to the formation of polymers with a molecular weight of about 64 kDa. This polymerisation inactivates the caspase and leads to the inhibition of cell death. In order to verify whether this hypothesis was true in our model system, we performed a western blot analysis of caspase-3 activation, after thapsigargin treatment, by means of an antibody able to recognize the processed forms of caspase-3. After 48 hours of treatment with 4 µg/ml of Thapsigargin, we observed the decrease of the 32 kDa signal, corresponding to the pro-caspase, in both cell line at the same extent. When we checked for the appearance of the two active forms of caspase-3 at 17 and 10 kDa, we could detect their appearance only in the SK-n-BE(2) cell line. On the other hand the TGA cell line show the appearance of a faint signal corresponding to the 10 kDa active form of caspase-3, but we detected the appearance of a strong signal at a molecular mass of about 34 kDa, as well as the increase of the signal at about 27 kDa. The molecular mass of these anti-caspase 3 positive bands suggests that they are polymers of the processed caspase-3 forms and that they are dependent on the thapsigargin induced activation of the TG2 cross-linking activity, even if slightly visible also in the SK-n-BE(2) cell line. These data are also supported by the measurement of the TG2 activity we performed upon thapsigargin treatment. In fact, upon thapsigargin treatment, we detected TG2 cross-linking activation in both cell lines even if more pronounced in the TGA cell line. The more evident formation of the caspase-3 oligomers, as well as the quite complete disappearence of the signal corresponding to the 17 kDa form, in the TGA cell line could explain the reduced sensitivity to cell death induction displayed by these cells. As a final verification we checked out if also the tunicamycin treatment might induce this polymerisation of the caspase-3 and then explain the slight protection observed in the TGA cell line. Even in this case we detected the polymerised form of caspase-3 in TGA cells only but at a minor extent in respect to the thapsigargin treatment. The data we obtained support the hypotesis that during ER-stress, the increase in cytosolic Ca2+ concentration is able to activate TG2as a cross-linking enzyme, which in turn acts on casp-3. The polymerisation of casp-3 inhibits its proteolytic activity thus protecting TG2 over-expressing cells from apoptosis.