Brian Bonish

Proteasome inhibitors trigger NOXA-mediated apoptosis in melanoma and myeloma cells

Patients with metastatic melanoma or multiple myeloma have a dismal prognosis because these aggressive malignancies resist conventional treatment. A promising new oncologic approach uses molecularly targeted therapeutics that overcomes apoptotic resistance and, at the same time, achieves tumor selectivity. The unexpected selectivity of proteasome inhibition for inducing apoptosis in cancer cells, but not in normal cells, prompted us to define the mechanism of action for this class of drugs, including Food and Drug Administration-approved bortezomib. In this report, five melanoma cell lines and a myeloma cell line are treated with three different proteasome inhibitors (MG-132, lactacystin, and bortezomib), and the mechanism underlying the apoptotic pathway is defined. Following exposure to proteasome inhibitors, effective killing of human melanoma and myeloma cells, but not of normal proliferating melanocytes, was shown to involve p53-independent induction of the BH3-only protein NOXA. Induction of NOXA at the protein level was preceded by enhanced transcription of NOXA mRNA. Engagement of mitochondrial-based apoptotic pathway involved release of cytochrome c, second mitochondria-derived activator of caspases, and apoptosis-inducing factor, accompanied by a proteolytic cascade with processing of caspases 9, 3, and 8 and poly(ADP)-ribose polymerase. Blocking NOXA induction using an antisense (but not control) oligonucleotide reduced the apoptotic response by 30% to 50%, indicating a NOXA-dependent component in the overall killing of melanoma cells. These results provide a novel mechanism for overcoming the apoptotic resistance of tumor cells, and validate agents triggering NOXA induction as potential selective cancer therapeutics for life-threatening malignancies such as melanoma and multiple myeloma.

Overexpression of CD1d by Keratinocytes in Psoriasis and CD1d-Dependent IFN-γ Production by NK-T Cells

The MHC class I-like protein CD1d is a nonpolymorphic molecule that plays a central role in development and activation of a subset of T cells that coexpress receptors used by NK cells (NK-T cells). Recently, T cells bearing NK receptors were identified in acute and chronic lesions of psoriasis. To determine whether NK-T cells could interact with epidermal cells, we examined the pattern of expression of CD1d in normal skin, psoriasis, and related skin disorders, using a panel of CD1d-specific mAbs. CD1d was expressed by keratinocytes in normal skin, although expression was at a relatively low level and was generally confined to upper level keratinocytes immediately beneath the lipid-rich stratum corneum. In contrast, there was overexpression of CD1d in chronic, active psoriatic plaques. CD1d could be rapidly induced on keratinocytes in normal skin by physical trauma that disrupted barrier function or by application of a potent contact-sensitizing agent. Keratinocytes displayed enhanced CD1d following exposure to IFN-γ. Combining CD1d-positive keratinocytes with human NK-T cell clones resulted in clustering of NK-T cells, and while no significant proliferation ensued, NK-T cells became activated to produce large amounts of IFN-γ. We conclude that CD1d can be expressed in a functionally active form by keratinocytes and is up-regulated in psoriasis and other inflammatory dermatoses. The ability of IFN-γ to enhance keratinocyte CD1d expression and the subsequent ability of CD1d-positive keratinocytes to activate NK-T cells to produce IFN-γ, could provide a mechanism that contributes to the pathogenesis of psoriasis and other skin disorders.

Innate Immune-Related Receptors in Normal and Psoriatic Skin

CONTEXT A precise role for the innate immune system in psoriasis remains to be determined. Surface receptors, including Toll-like receptors (TLRs) that recognize bacterial ligands and CD91, which recognizes heat shock proteins (HSPs), are implicated in both innate and adaptive immunity. OBJECTIVE Since skin is exposed to various exogenous stimuli, which can provoke or exacerbate psoriasis, we characterized expression and function of TLRs, CD91, and HSPs in normal and psoriatic skin. DESIGN A variety of skin-derived cells and blood-derived cells were analyzed both in vivo and in vitro; samples were obtained from 24 different individuals for innate immune-related receptor expression and function. By comparing and contrasting individuals with healthy skin and psoriatic patients, several specific differences were identified. RESULTS Immunohistochemistry-based expression profiling revealed TLR1 expression in epidermal dendritic cells (DCs) and dermal dendritic cells (DDCs) in normal skin, as well as in pre-psoriatic skin and psoriatic plaques, with enhanced basal layer keratinocyte (KC) expression in pre-psoriatic and psoriatic plaques compared with normal skin; TLR2 expression primarily by DDCs; and TLR4 expression by epidermal DCs and DDCs, with mid-epidermal-layer KCs displaying cell surface staining. No TLR9 or CD14 was detected on DCs or KCs, although psoriatic plaques contained CD14-positive macrophages. Analysis of psoriatic epidermis revealed HSPs 27, 60, and 70. Keratinocytes were CD91 negative, but CD91 was expressed by fibroblasts and DDCs in normal and pre-psoriatic skin, with prominent accumulation of CD91-positive DDCs in psoriatic plaques. Cultured KCs revealed no surface expression of TLR2, TLR4, TLR9, or CD91. Exposure of fibroblasts, but not KCs, to lipopolysaccharide or HSPs triggered nuclear factor (NF)-kappaB activation. Heat shock proteins did induce maturation of blood-derived DCs accompanied by increased interleukin-12 production and enhanced antigen-presenting function. CONCLUSIONS These data demonstrate distinctive patterns of innate immune-related receptors by specific subsets of cells in normal and psoriatic skin, suggesting functional roles for HSPs and DCs in psoriasis.

Characterization of a T cell line bearing natural killer receptors and capable of creating psoriasis in a SCID mouse model system

T cells bearing natural killer receptors (NKRs) such as CD94 and CD161 are present in psoriasis. These immunocytes express several receptors for both classical and non-classical class I MHC molecules. Whether T cells bearing NKRs in psoriatic lesions represent immunoregulatory versus pathogenic immunocytes or are just bystander cells is unclear. To address this issue, a CD94+/CD161+ T cell line was established from a psoriatic patient using IL-2/superantigens, and the interaction between NK-T cells and keratinocytes was characterized using in-vitro and in-vivo assays. Upon incubation between NK-T cells and CD1d positive keratinocytes, high levels of IFN-gamma and IL-13 were produced. Cytokine production was inhibited by a mAb against CD1d, implicating recognition of this surface molecule in the T cell response. Furthermore when this line was injected into pre-psoriatic skin engrafted onto a SCID mouse, a psoriatic plaque was created. The hyperplastic epidermal keratinocytes diffusely expressed CD1d, and were infiltrated by CD161+ T cells. RNase protection assay revealed predominantly IFN-gamma and IL-15 mRNAs, with barely detectable IL-13 mRNA in the acute lesion. These in-vivo findings demonstrated that this T cell line was pathogenic by creating a psoriatic plaque. The in-vitro results support a pathophysiologic role for interaction between T cells expressing NKRs and CD1d positive keratinocytes, with subsequent production of IFN-gamma. Upon injection in-vivo, the cytokine network produced was characterized by an immunological response polarized towards Th1 rather than Th2 cytokines. Thus, this pathogenic cell line provides evidence that T cells bearing NKRs can directly provoke a Th1 disease such as psoriasis.

Avoiding premature apoptosis of normal epidermal cells

DAVID LAWRENCE, ZAHRA SHAHROKH, SCOT MARSTERS, KIRSTEN ACHILLES, DANNY SHIH, BARBARA MOUNHO, KENNETH HILLAN, KLARA TOTPAL, LAURA DEFORGE, PETER SCHOW, JEFFREY HOOLEY, STEVE SHERWOOD, ROGER PAI, SUSAN LEUNG, LOLO KHAN, BRIAN GLINIAK, JEANINE BUSSIERE, CRAIG A. SMITH, STEPHEN S. STROM, SEAN KELLEY, JUDITH A. FOX, DEBORAH THOMAS & AVI ASHKENAZI Genentech Incorporated, South San Francisco, California, USA Immunex Corporation, Seattle, Washington, USA University of Pittsburgh, Pittsburgh, Pennsylvania, USA Email: aa@gene.com 1. Wiley, S.R. et al. Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity 3, 673–682 (1995). 2. Pitti, R.M. et al. Induction of apoptosis by Apo-2 Ligand, a new member of the tumor necrosis factor receptor family. J. Biol. Chem. 271, 12697–12690 (1996). 3. Ashkenazi, A. et al. Safety and anti-tumor activity of recombinant soluble Apo2 ligand. J. Clin. Invest. 104, 155–162 (1999). 4. Walczak, H. et al. Tumoricidal activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo. Nature Med. 5, 157–163 (1999). 5. Gliniak, B. & Le, T. Tumor necrosis factor-related apoptosis-inducing ligand’s antitmor activity in vivo is enhanced by the chemotherapeutic agent CPT-11. Cancer Res. 59, 6153–6158 (1999). 6. Roth, W. et al. Locoregional Apo2L/TRAIL eradicates intracranial human malignant glioma xenografts in athymic mice in the absence of neurotoxicity. Biochem. Biophys. Res. Commun. 265, 1999 (1999). 7. Chinnaiyan, A.M. et al. Combined effect of tumor necrosis factor-related apoptosis-inducing ligand and ionizing radiation in breast cancer therapy. Proc. Natl. Acad. Sci. USA 97, 1754–1759 (2000). 8. Jo, M. et al. Apoptosis induced in normal human hepatocytes by tumor necrosis factor-related apoptosis-inducing ligand. Nature Med. 6, 564–567 (2000). 9. Nagata, S. Steering anti-cancer drugs away from the TRAIL. Nature Med. 6, 502–503 (2000). 10. Hymowitz, S.G. et al. Triggering cell death: the crystal structure of Apo2L/TRAIL in a complex with death receptor 5. Molec. Cell 4, 563–571 (1999). 11. Hymowitz, S.G. et al. A unique zinc-binding site revealed by a high-resolution X-ray structure of homotrimeric Apo2L/TRAIL. Biochemistry 39, 633–640 (2000). 12. Bodmer, J.-L., Meier, P., Tschopp, J. & Schneider, P. Cysteine 230 is essential for the structure and activity of the cytotoxic ligand TRAIL. J. Biol. Chem. 275, 20632–20637 (2000). 13. Schneider, P. et al. Conversion of membranebound Fas(CD95) ligand to its soluble form is associated with downregulation of its proapoptotic activity and loss of liver toxicity. J. Exp. Med. 187, 1205–1213 (1998). 14. Nicoletti, I., Migliorati, G., Pagliacci, M.C., Grignani, F. & Riccardi, C. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J. Immunol. Methods 139, 271–279 (1991). 15. Kischkel, F.C. et al. Apo2L/TRAIL-dependent recruitment of endogenous FADD and caspase-8 to death receptors 4 and 5. Immunity 12, 611–620 (2000). 16. Ashkenazi, A. & Dixit, V.M. Apoptosis control by death and decoy receptors. Curr. Opin. Cell Biol. 11, 255–260 (1999).

The tumour necrosis factor-α inhibitor adalimumab rapidly reverses the decrease in epidermal Langerhans cell density in psoriatic plaques

Background  The pathophysiology of psoriasis is poorly understood, and the mechanism of action of biological agents interfering with tumour necrosis factor (TNF)‐α that improve psoriatic plaques is completely unknown.

Pathways involved in proliferating, senescent and immortalized keratinocyte cell death mediated by two different TRAIL preparations

Abstract: Properly regulated keratinocyte cell death is fundamentally important to maintain structural integrity and homeostatic function of epidermis. Moreover, from an oncological perspective, therapeutic approaches selectively targeting apoptosis of malignant cell types while sparing normal keratinocytes in surrounding skin is desirable. Apo2Ligand/tumor necrosis factor‐related apoptosis‐inducing ligand (Apo2L/TRAIL) has been observed to preferentially induce cytopathic effects on transformed/malignant cell types compared with their non‐neoplastic counterparts. In this report, two different biologically active preparations of Apo2L/TRAIL, a non‐tagged version, NT‐Apo2L/TRAIL, and a leucine zipper fusion protein, LZ‐Apo2L/TRAIL, were examined for their ability to trigger apoptosis in normal human keratinocytes, and in an immortalized cell line (HaCaT cells). Differences between these preparations were observed, including: NT‐Apo2L/TRAIL induced less keratinocyte apoptosis compared with LZ‐Apo2L/TRAIL; NT‐Apo2L/TRAIL also induced less apoptosis of HaCaT cells compared with LZ‐Apo2L/TRAIL; LZ‐Apo2L/TRAIL but not NT‐Apo2L/TRAIL induced cytotoxic effects when keratinocytes became growth arrested due to undergoing spontaneous replicative senescence – a biological state previously observed to be resistant to UV‐light‐induced apoptosis. Similarities between preparations included: an enhanced ability for both Apo2L/TRAIL preparations to kill a greater relative percentage of HaCaT cells compared with keratinocytes; enhanced cytotoxicity towards keratinocytes that had their NF‐B activity inhibited; a dependence of both Apo2L/TRAIL preparations on FADD and caspase activation; triggering of the same caspase cascades including caspase 8 and 3; and an ability to induce apoptosis even when HaCaT cells and keratinocytes were transduced to overexpress either Bcl‐2 or Bcl‐xL (survival factors that reduce susceptibility to UV‐light‐induced apoptosis). These results indicate that while both preparations of Apo2L/TRAIL possess biological activity, there are important differences as regards their ability to induce apoptosis in normal and immortalized keratinocytes. Moreover, the death receptor pathway triggered by LZ‐Apo2L/TRAIL can overcome the apoptotic resistance normally observed in response to UV‐light mediated by Bcl‐2/Bcl‐xL, as well as by the state of cellular senescence. Unraveling the molecular basis for these differential biological effects may reveal a new strategic role for these death receptor/ligands linked to apoptosis in maintaining the dynamic balance of keratinocyte proliferation, differentiation, and cell death necessary to achieve a homeostatic thickness and function of normal skin. In addition, it may be possible to utilize these Apo2L/TRAIL preparations for the treatment of various sun‐induced skin cancers as they can differentially trigger apoptosis of transformed keratinocytes, or keratinocytes with abnormal NF‐κB signaling, while sparing adjacent normal keratinocytes.

Expansion of vitiligo lesions is associated with reduced epidermal CDw60 expression and increased expression of HLA-DR in perilesional skin

Background  Detection of CDw60 in skin is representative of ganglioside D3 expression. This ganglioside is expressed primarily by melanocytes, and is of interest as a membrane antigen targeted by immunotherapy for melanoma patients. Expression of CDw60 by keratinocytes is defined by the presence of T‐helper cell (Th)1 vs. Th2 cytokines, and can serve as a sentinel molecule to characterize an ongoing skin immune response.

Phenotype and proliferation characteristics of cultured spindle-shaped cells obtained from normal human skin and lesions of dermatofibroma, Kaposi's sarcoma, and dermatofibrosarcoma protuberans: a comparison with fibroblast and endothelial cells of the dermis

Normal human dermis contains mesenchymal cells that are generally referred to as fibroblasts. However the relationships between fibroblasts and endothelial cells with respect to the types of spindle-shaped cells that are present in cultures obtained from tumor bearing-skin is unclear. To explore the potential heterogeneity amongst dermal-derived cells that grow in culture with a spindle-shaped morphology, we compared the immunophenotype and growth characteristics of several types of cells. Besides dermal fibroblasts and microvascular endothelial cells derived from normal adult skin, we also studied large vessel-derived endothelial cells, and spindle-shaped cells derived from three different tumor-bearing dermal-based neoplasms. Kaposis sarcoma (KS), dermatofibroma (DF), and dermatofibrosarcoma protuberans (DFSP). A broad panel of eight different antibodies were used to immunophenotype the multi-passaged cultured cells. Spindle-shaped cells from all three neoplasms could be distinguished from the normal skin derived fibroblasts by their constitutive expression of factor XIIIa, and the gamma-interferon induced expression of VCAM-1. All seven types of cultured cells stained positive for s-actin and proline-4-hydroxylase, and none of the cells expressed CD34. Both large and small-vessel derived endothelial cells expressed factor VIII, ELAM-1, and VCAM-1. Using two different types of growth media, significant differences were also observed amongst these cultured cell types. Spindle-shaped cells from DFSP did not grow in DMEM containing 10% fetal bovine serum (DMEM-FBS); but they proliferated in KS cell growth medium (KSGM). Spindle-shaped cells from DF grew best in KSGM, but not in DMEM-FBS. KS tumor cells grew well in KSGM, but not in DMEM-FBS. Fibroblasts proliferated in DMEM-FBS, but failed to grow in KSGM; and even when pre-treated with conditioned medium from a transformed KS cell line (i.e. SLK cells), no fibroblast proliferation could be induced in KSGM. These results indicate that KS cell line (i.e. SLK cells), no fibroblast proliferation could be induced in KSGM. These results indicate that even though dermal-derived cells can have an identical spindle-shape by light microscopy, significant heterogeneity can be defined amongst such cells from normal and tumor-bearing human skin. Having established culture conditions to propagate these different cell types and phenotypic criteria to distinguish them from one another, will provide new research opportunities to explore the function and ontogeny of the diverse mesenchymal cells that take on a spindle-shaped morphology in culture.

Role for Id‐1 in immunobiology of normal keratinocytes and in basal cell carcinoma

Abstract:  It has been established that Id proteins can block the basic helix–loop–helix (HLH) transcription factors, thereby impacting the onset of senescence in keratinocytes, as well as influencing tumorigenesis involving squamous cell carcinomas. However, the ability of Id‐1 to influence the immunologic response of epithelial cells to cytokines implicated in cutaneous oncology such as gamma interferon (IFN‐γ) has not been determined. Using a whole population of human keratinocytes infected with a retrovirus to induce over‐expression of Id‐1, the influence on early differentiation of rapidly proliferating keratinocytes was assessed, as was the response to IFN‐γ. While induction of involucrin, a marker of early differentiation, was not altered in Id‐1 overexpressing keratinocytes, the IFN‐γ mediated increase in intercellular adhesion molecule‐1 (ICAM‐1) and HLA‐DR was reduced. No change in constitutive or inducible levels of MHC class I antigen, CD95 (Fas antigen) or LFA‐3 (CD58) was observed in this system. Immunostaining and Western blot analysis revealed over‐expression of Id‐1 in basal cell carcinomas (BCCs). These tumors not only strongly and diffusely expressed Id‐1, but were also characterized by reduced ICAM‐1 and HLA‐DR expression. Thus, dysregulated Id‐1 may not only contribute to delaying the senescence program in keratinocytes, it may also contribute to the escape of the relatively undifferentiated tumor cells in BCC from immune surveillance.