Marco Cippitelli

ATM-ATR-dependent up-regulation of DNAM-1 and NKG2D ligands on multiple myeloma cells by therapeutic agents results in enhanced NK-cell susceptibility and is associated with a senescent phenotype.

There is much evidence to support a role for natural killer (NK) cells in controlling the progression of multiple myeloma (MM), a malignancy characterized by an abnormal plasma cell proliferation in the bone marrow (BM). Induction of DNA damage response has been recently shown capable of enhancing NKG2D ligand (NKG2DL) expression, but nothing is known about DNAM-1 ligand (DNAM-1L) regulation. In this study, we show that myeloma cells treated with low doses of therapeutic agents commonly used in the management of patients with MM, such as doxorubicin, melphalan, and bortezomib, up-regulate DNAM-1 and NKG2D ligands. Accordingly, therapeutic drug treatment of MM cells increases NK-cell degranulation, the NKG2D and DNAM-1 receptors being the major triggering molecules. Similar data were also obtained using ex vivo primary plasma cells derived from MM patients. Drug-induced DNAM-1 and NKG2D ligand expression was abolished after treatment with the ATM (ataxia telangiectasia mutated) and ATR (ATM- and RAD3-related) pharmacologic inhibitors caffeine and KU-55933, and was preferentially associated with senescent cells arrested in the G2 phase of the cell cycle. Altogether, our findings have identified a common pathway that can trigger the up-regulation of different NK cell-activating ligands and suggest that NK cells represent an immunosurveillance mechanism toward cells undergoing stress-induced senescent programs.

Vitamin D3: a transcriptional modulator of the interferon-γ gene

1α,25‐Dihydroxyvitamin D3 [1225‐(OH)2D3] exerts several effects on the immune system, by regulating lymphocyte proliferation, differentiation of monocytes and secretion of cytokines as IL‐2, granulocyte‐macrophage colony‐stimulating factor and IFN‐γ in T cells. Here, we analyze the effect of 1,25‐(OH)2D3 on IFN‐γ gene transcription. Transient transfection assays in Jurkat T cells indicate that activation of the IFN‐γ promoter is down‐regulated by 1,25‐(OH)2D3. This effect is enhanced by retinoid X receptor (RXR), and a functional vitamin D3 receptor (VDR) DNA‐binding domain in necessary for repression. We delineated two important promoter regions mainly involved in this modulation. The first of these is situated at the level of a promoter‐silencer previously characterized and binds the heterodimer VDR‐RXR in electrophoretic mobility shift assay. Residual negative regulation was also detected at the level of the promoter fragment – 108 to + 64 bp from the transcription start site and, surprisingly, the activity of the IFN‐γ enhancer from – 108 to – 36 bp in the context of a heterologous promoter was not affected by 1,25‐(OH)2D3. Moreover, binding activity for VDR‐RXR has been detected in the IFN‐γ minimal promoter, suggesting a possible mechanism of interference with transcription initiation/progression. The overall data indicate that direct modulation of the IFN‐γ promoter activity is one of the possible mechanisms involved in the repressive effect of 1,25‐(OH)2D3 on IFN‐γ gene expression.

bcl‐2 over‐expression enhances NF‐κB activity and induces mmp‐9 transcription in human MCF7ADR breast‐cancer cells

bcl‐2 expression is often associated with poor prognosis in several types of tumors; however, the role of this molecule in breast cancer is still controversial. We found earlier that over‐expression of bcl‐2 in a human breast‐cancer cell line (MCF7ADR) enhances its tumorigenicity and metastatic potential by inducing metastasis‐associated properties such as increased secretion of the matrix metalloproteinase‐9 (mmp‐9). In the present study, we investigated the effect of bcl‐2 over‐expression on the activity of the transcription factor NF‐κB, an important regulator of genes involved in tumor progression and invasion. Transient transfection experiments indicate that over‐expression of bcl‐2 in the MCF7ADR cell line, enhances NF‐κB‐dependent transcriptional activity. Mobility‐shift analysis revealed an increase of NF‐κB DNA‐binding in bcl‐2‐over‐expressing clones that correlated with lower levels of the NF‐κB cytoplasmic inhibitor IκBα. Moreover, point mutations of 2 highly conserved residues within the BH1 and BH2 domains that abrogate the interaction of bcl‐2 with bax, or deletion of the N‐terminal BH4 domain, completely eliminate the ability of this molecule to up‐regulate NF‐κB‐dependent transactivation. Since mmp‐9 is a NF‐κB‐regulated gene, we also investigated whether bcl‐2 over‐expression up‐regulated mmp‐9 transcription. We found that induction of mmp‐9 mRNA correlates with the activation of an mmp‐9‐promoter‐reporter‐gene construct in transient transfection assay, and a mutation of the (−600)mmp‐9‐NF‐κB binding element abolishes this effect. The overall data indicate that bcl‐2‐mediated regulation of NF‐κB‐transcription‐factor activity may represent an important mechanism for the promotion of malignant behavior in MCF‐7ADR cells. Int. J. Cancer 86:188–196, 2000.

Negative Regulation of CD95 Ligand Gene Expression by Vitamin D3 in T Lymphocytes

Fas (APO-1/CD95) and its ligand (FasL/CD95L) are cell surface proteins whose interaction activates apoptosis of Fas-expressing targets. In T lymphocytes, the Fas/FasL system regulates activation-induced cell death, a fundamental mechanism for negative selection of immature T cells in the thymus and for maintenance of peripheral tolerance. Aberrant expression of Fas and FasL has also been implicated in diseases in which the lymphocyte homeostasis is compromised, and several studies have described the pathogenic functions of Fas and FasL in vivo, particularly in the induction/regulation of organ-specific autoimmune diseases. The 1,25(OH)2D3 is a secosteroid hormone that activates the nuclear receptor vitamin D3 receptor (VDR), whose immunosuppressive activities have been well studied in different models of autoimmune disease and in experimental organ transplantation. We and others have recently described the molecular mechanisms responsible for the negative regulation of the IFN-γ and IL-12 genes by 1,25(OH)2D3 in activated T lymphocytes and macrophages/dendritic cells. In this study, we describe the effect of 1,25(OH)2D3 on the activation of the fasL gene in T lymphocytes. We show that 1,25(OH)2D3 inhibits activation-induced cell death, fasL mRNA expression, and that 1,25(OH)2D3-activated VDR represses fasL promoter activity by a mechanism dependent on the presence of a functional VDR DNA-binding domain and ligand-dependent transcriptional activation domain (AF-2). Moreover, we identified a minimal region of the promoter containing the transcription start site and a noncanonical c-Myc-binding element, which mediates this repression. These results place FasL as a novel target for the immunoregulatory activities of 1,25(OH)2D3, and confirm the interest for a possible pharmacological use of this molecule and its derivatives.

The DNA damage response: a common pathway in the regulation of NKG2D and DNAM-1 ligand expression in normal, infected, and cancer cells

NKG2D and DNAM-1 are two activating receptors, present on the surface of NK cells and other cells of the immune system. Their ligands – MICA, MICB, ULBP1-6 for NKG2D, PVR/CD155 and Nectin-2/CD112 for DNAM-1 – can be constitutively expressed at low levels in some normal cells, but they are more often defined as “stress-induced,” since different stimuli can positively regulate their expression. In this review, we describe the molecular mechanisms involved in the up-regulation of NKG2D and DNAM-1 ligands under different physiological and pathological “stress” conditions, including mitosis, viral infections, and cancer. We will focus on the DNA damage response, as recent advances in the field have uncovered its important role as a common signaling pathway in the regulation of both NKG2D and DNAM-1 ligand expression in response to very diverse conditions and stimuli.

Heat Shock Protein-90 Inhibitors Increase MHC Class I-Related Chain A and B Ligand Expression on Multiple Myeloma Cells and Their Ability to Trigger NK Cell Degranulation

Modulation of the host immune system represents a promising therapeutic approach against cancer, including multiple myeloma. Recent findings indicate that the NK group 2D (NKG2D)- and DNAX accessory molecule-1 (DNAM-1)-activating receptors play a prominent role in tumor recognition and elimination by cytotoxic lymphocytes, suggesting that the levels of NKG2D and DNAM-1 ligand expression on tumor cells may be a critical factor to improve the immune response against cancer. In this study, we tested the effect of 17-allylaminogeldanamycin and radicicol, drugs targeting the heat shock protein-90 (HSP-90) chaperone protein and displaying antimyeloma activity, on the expression of NKG2D and DNAM-1 ligands in human myeloma cell lines. We demonstrate that HSP-90 inhibitors are able to up-regulate both MHC class I chain-related (MIC) A and MICB protein surface and mRNA expression in human myeloma cell lines, without any significant effect on the basal expression of the DNAM-1 ligand poliovirus receptor CD155, or induction of nectin-2 and UL16-binding proteins. Activation of the transcription factor heat shock factor-1 by HSP-90 inhibitors is essential for the up-regulation of MICA/MICB expression and knockdown of heat shock factor-1 using small hairpin RNA interference blocks this effect. Moreover, in vitro and in vivo binding of heat shock factor-1 to MICA and MICB promoters indicates that it may enhance NKG2D ligand expression at the transcriptional level. Finally, exposure to HSP-90 inhibitors renders myeloma cells more efficient to activate NK cell degranulation and a blocking Ab specific for NKG2D significantly reduces this effect. Thus, these results provide evidence that targeting NKG2D ligands expression may be an additional mechanism supporting the antimyeloma activity of HSP-90 inhibitors and suggest their possible immunotherapeutic value.

relA over-expression reduces tumorigenicity and activates apoptosis in human cancer cells.

We previously demonstrated that bcl-2 over-expression increases the malignant behaviour of the MCF7 ADR human breast cancer cell line and enhances nuclear factor-kappa B (NF-kB) transcriptional activity. Here, we investigated the direct effect of increased NF-kB activity on the tumorigenicity of MCF7 ADR cells by over-expressing the NF-kB subunit relA/p65. Surprisingly, our results demonstrated that over-expression of relA determines a considerable reduction of the tumorigenic ability in nude mice as indicated by the tumour take and the median time of tumour appearance. In vitro studies also evidenced a reduced cell proliferation and the activation of the apoptotic programme after relA over-expression. Apoptosis was associated with the production of reactive oxygen species, and the cleavage of the specific substrate Poly-ADP-ribose-polymerrase. Our data indicate that there is no general role for NF-kB in the regulation of apoptosis and tumorigenicity. In fact, even though inhibiting NF-kB activity has been reported to be lethal to tumour cells, our findings clearly suggest that an over-induction of nuclear NF-kB activity may produce the same effect.

Reactive Oxygen Species– and DNA Damage Response–Dependent NK Cell Activating Ligand Upregulation Occurs at Transcriptional Levels and Requires the Transcriptional Factor E2F1

Increasing evidence indicates that cancer cell stress induced by chemotherapeutic agents promote antitumor immune responses and contribute to their full clinical efficacy. In this article, we identify the signaling events underlying chemotherapy-induced NKG2D and DNAM-1 ligand expression on multiple myeloma (MM) cells. Our findings indicate that sublethal doses of doxorubicin and melphalan initiate a DNA damage response (DDR) controlling ligand upregulation on MM cell lines and patient-derived malignant plasma cells in Chk1/2-dependent and p53-independent manner. Drug-induced MICA and PVR gene expression are transcriptionally regulated and involve DDR-dependent E2F1 transcription factor activity. We also describe the involvement of changes in the redox state in the control of DDR-dependent upregulation of ligand surface expression and gene transcriptional activity by using the antioxidant agent N-acetyl-l-cysteine. Finally, in accordance with much evidence indicating that DDR and oxidative stress are major determinants of cellular senescence, we found that redox-dependent DDR activation upon chemotherapeutic treatment is critical for MM cell entry in premature senescence and is required for the preferential ligand upregulation on senescent cells, which are preferentially killed by NK cells and trigger potent IFN-γ production. We propose immunogenic senescence as a mechanism that promotes the clearance of drug-treated tumor cells by innate effector lymphocytes, including NK cells.

Hyperthermia Enhances CD95-Ligand Gene Expression in T Lymphocytes

Hyperthermia represents an interesting therapeutic strategy for the treatment of tumors. Moreover, it is able to regulate several aspects of the immune response. Fas (APO-1/CD95) and its ligand (FasL) are cell surface proteins whose interaction activates apoptosis of Fas-expressing targets. In T cells, the Fas-Fas-L system regulates activation-induced cell death, is implicated in diseases in which lymphocyte homeostasis is compromised, and plays an important role during cytotoxic and regulatory actions mediated by these cells. In this study we describe the effect of hyperthermia on activation of the fas-L gene in T lymphocytes. We show that hyperthermic treatment enhances Fas-L-mediated cytotoxicity, fas-L mRNA expression, and fas-L promoter activity in activated T cell lines. Our data indicate that hyperthermia enhances the transcriptional activity of AP-1 and NF-κB in activated T cells, and this correlates with an increased expression/nuclear translocation of these transcription factors. Moreover, we found that heat shock factor-1 is a transactivator of fas-L promoter in activated T cells, and the overexpression of a dominant negative form of heat shock factor-1 may attenuate the effect of hyperthermia on fas-L promoter activity. Furthermore, overexpression of dominant negative mutants of protein kinase Cε (PKCε) and PKCθ partially inhibited the promoter activation and, more importantly, could significantly reduce the enhancement mediated by hyperthermia, indicating that modulation of PKC activity may play an important role in this regulation. These results add novel information on the immunomodulatory action of heat, in particular in the context of its possible use as an adjuvant therapeutic strategy to consider for the treatment of cancer.

Inhibition of Glycogen Synthase Kinase-3 Increases NKG2D Ligand MICA Expression and Sensitivity to NK Cell–Mediated Cytotoxicity in Multiple Myeloma Cells: Role of STAT3

Engagement of NKG2D and DNAX accessory molecule-1 (DNAM-1) receptors on lymphocytes plays an important role for anticancer response and represents an interesting therapeutic target for pharmacological modulation. In this study, we investigated the effect of inhibitors targeting the glycogen synthase kinase-3 (GSK3) on the expression of NKG2D and DNAM-1 ligands in multiple myeloma (MM) cells. GSK3 is a pleiotropic serine–threonine kinase point of convergence of numerous cell-signaling pathways, able to regulate the proliferation and survival of cancer cells, including MM. We found that inhibition of GSK3 upregulates both MICA protein surface and mRNA expression in MM cells, with little or no effects on the basal expression of the MICB and DNAM-1 ligand poliovirus receptor/CD155. Moreover, exposure to GSK3 inhibitors renders myeloma cells more efficient to activate NK cell degranulation and to enhance the ability of myeloma cells to trigger NK cell–mediated cytotoxicity. We could exclude that increased expression of β-catenin or activation of the heat shock factor-1 (transcription factors inhibited by active GSK3) is involved in the upregulation of MICA expression, by using RNA interference or viral transduction of constitutive active forms. On the contrary, inhibition of GSK3 correlated with a downregulation of STAT3 activation, a negative regulator of MICA transcription. Both Tyr705 phosphorylation and binding of STAT3 on MICA promoter are reduced by GSK3 inhibitors; in addition, overexpression of a constitutively active form of STAT3 significantly inhibits MICA upregulation. Thus, we provide evidence that regulation of the NKG2D-ligand MICA expression may represent an additional immune-mediated mechanism supporting the antimyeloma activity of GSK3 inhibitors.